Methods and compositions for treating a fibrotic disease

ABSTRACT

The present invention provides compositions and methods for the treatment of diseases and disorders. In some embodiments the compositions and methods involve administration of a microparticle, or composition thereof that includes an antifibrotic.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a 35 U.S.C. § 371 National Phase Entry Applicationof International Application No. PCT/US2021/033606 filed May 21, 2021,which designates the U.S. and claims benefit under 35 U.S.C. § 119(e) ofU.S. Provisional Application No. 63/028,961 filed May 22, 2020, thecontents of which are incorporated herein by reference in theirentireties.

SEQUENCE LISTING

[0001.1] The instant application contains a Sequence Listing which hasbeen submitted electronically in ASCII format and is hereby incorporatedby reference in its entirety. Said ASCII copy, created on Jul. 6, 2021,is named 701586-097700USPT_SL.txt and is 22,186 bytes in size.

TECHNICAL FIELD

The technology described herein relates to methods and compositions fortreating a fibrotic disease.

BACKGROUND

Joint stiffness is a significant public health issue with currenttreatment options providing varied and limited outcomes. Joint stiffnesscan affect any joint in the body, such as a shoulder joint, an elbowjoint, a wrist joint, a finger joint, a hip joint, a knee joint, anankle joint, a toe joint, the spine and the jaw A shoulder joint isoften affected by joint stiffness, which is also termed a shouldercontracture, and is also known as “frozen shoulder”.

Shoulder contracture is considered a self-limiting disease, but recoveryis protracted and arduous, with a significant number of patients neverregaining full ROM. The reported outcomes of conservative therapy (i.e.,physical therapy) vary considerably and are highly dependent on how theyare measured (Neviaser A.S. and Neviaser R.J., J. Am. Acad. Orthop.Surg. 2011, 19(9):536-42). Results tend to be more favorable withsubjective outcome measures than with objective outcome measures. In onestudy, 90% of patients treated with minimal therapy reportedsatisfaction with their shoulder function (Griggs S.M. et al., J. BoneJoint Surg. Am. 2000, 82-A(10): 1398-407). However, another that usedobjective outcomes reported residual pain in 50% of patients and motiondeficit in 60% (Shaffer B. et al., J. Bone Joint Surg. Am.1992;74(5):738-46). Mild to moderate symptoms can persist after 4.4years following symptom onset of shoulder contracture. For thoseexperiencing severe disease, such functional impairment interferes withdaily activities and work-related responsibilities (Hand C. et al.,Journal of Shoulder and Elbow Surgery 2008, 17(2):231-6). When patientsdo not respond to conservative management, other treatment options areavailable. Operative intervention in the form of manipulation underanesthesia may restore motion and decrease pain, but it has beenassociated with complications such as fracture, tendon rupture, andneurologic injury (Castellarin G. et al., Archives of Physical Medicineand Rehabilitation 2004, 85(8): 1236-40; Hsu S.Y. and Chan K.M.,International Orthopaedics, 1991, 15(2):79-83; Parker R.D. et al.,Orthopedics, 1989, 12(7):989-90). There are reports that manipulation orcapsular release do not offer reliable and consistent results (ShafferB. et al., J. Bone Joint Surg. Am. 1992, 74(5):738-46; Ryans I. et al.,Rheumatology 2005, 44(4):529-35). Accordingly, a more effective andconsistent therapy for joint stiffness is needed.

See also, PCT Application No US2017/055799; PCT Application NoUS14/380163; and Disseration defense of William Blessing dated Aug. 26,2019; the contents of which are incorporated herein by reference intheir entirties.

SUMMARY

One aspect described herein provides a formulation comprisingmicroparticles comprising an aliphatic polyester and an antifibroticagent, wherein (i) said microparticles have a diameter of 1-100 µm; (ii)the antifibrotic agent is relaxin and is present in an amount that is0.01-33% of total mass; (iii) said aliphatic polyester is of molecularweight 10,000-200,000 Daltons; or (iv) the microparticles furthercomprise a vinyl polymer.

In one embodiment of any aspect herein, said antifibrotic agent is anagonist of the receptor RXFP1.

In one embodiment of any aspect herein, said antifibrotic agent is humanrelaxin-2 or an analog or variant.

In one embodiment of any aspect herein, the aliphatic polyester ispoly-lactide-co-glycolide.

In one embodiment of any aspect herein, the aliphatic polyester ispolycaprolactone.

In one embodiment of any aspect herein, said aliphatic polyester isterminated by an ester functional group, an alkyl-ester functionalgroup, an amine functional group, an isocyanate functional group, anisothiocyanate functional group, a benzoyl fluoride functional group, amaleimide functional group, an iodoacetamide functional group, a2-thiopyridine functional groups, a 3-arylpropiolonitrile functionalgroup, a diazonium salt, an aldehyde, a ketone, an azide, an alkyne, acyclooctyne, a phosphine or a carboxylic acid functional group.

In one embodiment of any aspect herein, said formulation comprises avinyl polymer that is poly(vinyl alcohol) or poly(pyrrolidone).

In one embodiment of any aspect herein, said formulation comprises avinyl polymer that is of molecular weight 10,000-200,000 Daltons.

In one embodiment of any aspect herein, the diameter of saidmicroparticles is 1-75 µm; or 1-50 µm; or 5-50 µm; or 25-50 µm; or 30-50µm; or 40-50 µm.

In one embodiment of any aspect herein, said aliphatic polyester ispoly-lactide-co-glycolide with a molar ratio of 15:85 - 25:75,lactide:glycolide.

In one embodiment of any aspect herein, the formulation comprises thevinyl polymer in an amount of 0.01-1.0% of total mass.

In one embodiment of any aspect herein, said antifibrotic agent is0.005-5% of the total formulation mass.

In one embodiment of any aspect herein, said antifibrotic agent is arelaxin.

In one embodiment of any aspect herein, said formulation comprisesmicroparticles suspended in a liquid solution.

In one embodiment of any aspect herein, said formulation comprisesmicroparticles suspended in a sodium chloride liquid solution or asodium carboxymethylcellulose solution.

In one embodiment of any aspect herein, said formulation is a sustainedrelease formulation.

In one embodiment of any aspect herein, said formulation is a sustainedrelease formulation and wherein the antifibrotic agent is released overan extended period of time.

In one embodiment of any aspect herein, said formulation is a sustainedrelease formulation wherein the antifibrotic agent is released over anextended period of least 1 day.

In one embodiment of any aspect herein, the formulation is formulatedfor administration via inhalation as an aerosol, administration viaintra-articular injection or administration via intramuscular injection.

In one embodiment of any aspect herein, the formulation is administeredto the subject such that the antifibrotic agent is administered to asubject at a dose between 1-2000 µg/kg body weight.

In some embodiment of any aspect herein, the formulation as providedherein is suspended in a liquid solution. In some embodiments themicroparticle formulation is suspended in a liquid solution wherein themicroparticle is about 0.0001-0.001% of the total solution; or is about0.001-0.01% of the total solution; or about 0.01-0.05% of the totalsolution; or is about 0.05-0.1% of the total solution; or is about0.1-1% of the total solution; or is about 1-10% of the total solution;or is about 10-20% of the total solution; or is about 20-30% of thetotal solution; or is about 30-50% of the total solution; or is about50-75% of the total solution; or is about 75-90% of the total solution.In some embodiments the aforementioned percentages are by weight, inother embodiments, the aforementioned percentages are by volume.

In one embodiment of any aspect herein, the formulation comprisesmicroparticles having an aliphatic polyester and an antifibrotic agent,wherein the microparticles have a diameter of 1-100 µm.

In one embodiment of any aspect herein, the formulation comprisesmicroparticles having an aliphatic polyester and an antifibrotic agent,wherein the antifibrotic agent is relaxin and is present in an amountthat is 0.01-33% of total mass.

In one embodiment of any aspect herein, the formulation comprisesmicroparticles having an aliphatic polyester and an antifibrotic agent,wherein said aliphatic polyester is of molecular weight 10,000-200,000daltons.

In one embodiment of any aspect herein, the formulation comprisesmicroparticles having an aliphatic polyester, a vinyl polymer and anantifibrotic agent.

In one embodiment of any aspect herein, the formulation comprisesmicroparticles having an aliphatic polyester, a vinyl polymer and anantifibrotic agent, wherein said microparticles have a diameter of 1-100µm.

A method of treating a fibrotic disease, the method comprisingadministering to a subject in need thereof any formulation describedherein.

A method, said method comprising identifying a subject diagnosed withone or more diseases selected from the group of diseases listed in Table1 or Table 2 and administering any formulation described herein to thesubject.

In one embodiment of any aspect herein, the disease is selected from thegroup consisting of Duchenne Muscular Dystrophy, Becker MuscularDystrophy, Spinal Muscular Atrophy (Type I, II, III, or IV), CerebralPalsy, Stroke, Traumatic Brain Injury, peripheral nerve injury,Arthrogryposis Multiplex Congenita, fibrosis of the humeroradial joint,fibrosis of the humeroulnar joint, fibrosis of the glenohumeral joint,fibrosis of the tibiofemoral joint, fibrosis of the acetabulofemoraljoint, fibrosis of the talocrural joint, fibrosis of thetemporomandibular joint, fibrosis of the metacarpophalangeal joint,fibrosis of the metatarsophalangeal joint, fibrosis of theperi-articular musculature, cellulite and interstitial lung disease.

In one embodiment of any aspect herein, said administering is viainhalation as an aerosol, via intra-articular injection, viaperi-articular injection, via intramuscular injection, via perimuscularinjection, via intradermal injection., via subcutaneous injection, viaintracapsular injection, via pericapsular injection, viaintraligamentous injection, via periligamentous injection, viaintratendinous injection, via peritendinous injection, viaintramusculotendionous injection, via perimusculotendinous injection,via intraosteotendinous injection, via periosteotendinous injection.

In one embodiment of any aspect herein, the disease is Duchene’smuscular dystrophy and said administering is via intramuscularinjection; the disease is Duchene’s muscular dystrophyand saidadministering is via intraarticular injection; the disease is Becker’smuscular dystrophy and said administering is via intramuscularinjection; the disease is Becker’s muscular dystrophy and saidadministering is via intraarticular injection; the disease is SpinalMuscular Dystrophy and said administering is via intramuscularinjection; the disease is Spinal Muscular Dystrophy and saidadministering is via intraarticular injection; the disease isArthrogryposis Multiplex Congenita and said administering is viaintramuscular injection; the disease is Arthrogryposis MultiplexCongenita and said administering is via intraarticular injection; thedisease is Cerebral Palsy and said administering is via intramuscularinjection; the disease is Cerebral Palsy and said administering is viaintraarticular injection; the disease is Stroke and said administeringis via intramuscular injection; the disease is Stroke and saidadministering is via intraarticular injection; the disease is TraumaticBrain Injury and said administering is via intramuscular injection; thedisease is Traumatic Brain Injury and said administering is viaintraarticular injection; the disease is peripheral nerve injury andsaid administering is via intramuscular injection; the disease isperipheral nerve injury and said administering is via intraarticularinjection.

In one embodiment of any aspect herein, microparticles are of sizesbetween 1 um-10 um and said administering is via inhalation as anaerosol; the microparticles are of sizes between 20 um-100 um and saidadministering is via intramuscular injection; the microparticles are ofsizes between 5 um-50 um and said administering is via intraarticularinjection.

In one embodiment of any aspect herein, the disease is interstitial lungdisease, the diameter of the microparticle is 1-10 um, and saidadministering is via inhalation as an aerosol; the disease is Duchene’sMuscular Dystrophy, the diameter of the microparticle is 10-30 um, andsaid administering is via intraarticular injection; the disease isDuchene’s Muscular Dystrophy, the diameter of the microparticle is 25-50um, and said administering is via intraarticular injection; the diseaseis Duchene’s Muscular Dystrophy, the diameter of the microparticle is10-30 um, and said administering is via intramuscular injection; thedisease is Duchene’s Muscular Dystrophy, the diameter of themicroparticle is 25-50 um, and said administering is via intramuscularinjection; the disease is Spinal Muscular Atrophy, the diameter of themicroparticle is 10-30 um, and said administering is via intraarticularinjection; the disease is Spinal Muscular Atrophy, the diameter of themicroparticle is 25-50 um, and said administering is via intraarticularinjection; the disease is Spinal Muscular Atrophy, the diameter of themicroparticle is 10-30 um, and said administering is via intramuscularinjection; the disease is Spinal Muscular Atrophy, the diameter of themicroparticle is 25-50 um, and said administering is via intramuscularinjection; the disease is joint arthrofibrosis, the diameter of themicroparticle is 10-30 um, and said administering is via intraarticularinjection; the disease is joint arthrofibrosis, the diameter of themicroparticle is 25-50 um, and said administering is via intraarticularinjection, ; the disease is joint Cerebral Palsy, the diameter of themicroparticle is 10-30um, and said administering is via intraarticularinjection; the disease is joint Cerebral Palsy, the diameter of themicroparticle is 25-50 um, and said administering is via intraarticularinjection; the disease is joint Stroke, the diameter of themicroparticle is 10-30 um, and said administering is via intraarticularinjection; the disease is joint Stroke, the diameter of themicroparticle is 25-50 um, and said administering is via intraarticularinjection; the disease is Traumatic Brain Injury, the diameter of themicroparticle is 10-30 um, and said administering is via intraarticularinjection; the disease is Traumatic Brain Injury, the diameter of themicroparticle is 25-50 um, and said administering is via intraarticularinjection; the disease is peripheral nerve injury, the diameter of themicroparticle is 10-30 um, and said administering is via intraarticularinjection; the disease is peripheral nerve injury, the diameter of themicroparticle is 25-50 um, and said administering is via intraarticularinjection.

In one embodiment of any aspect herein, the formulation is administeredto the subject such that the antifibrotic agent is administered to asubject at a dose between 1-2000 µg/kg body weight.

Definitions

For convenience, the meaning of some terms and phrases used in theinvention, examples, and appended claims, are provided. Unless statedotherwise, or implicit from context, the following terms and phrasesinclude the meanings provided below. The definitions are provided to aidin describing particular embodiments, and are not intended to limit theclaimed technology, because the scope of the technology is limited onlyby the claims. Unless otherwise defined, all technical and scientificterms used herein have the same meaning as commonly understood by one ofordinary skill in the art to which this technology belongs. If there isan apparent discrepancy between the usage of a term in the art and itsdefinition provided herein, the definition provided within thespecification shall prevail.

As used herein, an “effective amount,” is intended to include the amountof the agent e.g., relaxin or an analog, a fragment or a variantthereof, that, when administered to a subject via a depot having astiffened joint, is sufficient to affect treatment of the stiffenedjoint (e.g., by diminishing, ameliorating or maintaining the stiffenedjoint or one or more symptoms of the stiffened joint). The “effectiveamount” may vary depending on the sequence of the agent, how the agentis administered, the severity of the joint stiffness and the history,age, weight, family history, genetic makeup, the types of preceding orconcomitant treatments, if any, and other individual characteristics ofthe subject to be treated.

The term “effective amount,” as used herein, is also intended to includethe amount of agent e.g., relaxin or an analog, a fragment or a variantthereof, that, when administered to a subject in a depot with astiffened joint, and either currently or not yet experiencing ordisplaying symptoms of the stiffened joint, such as pain on movement orrestriction of the movement or range of movement of the joint affectedby the joint stiffness, and/or a subject at risk of developing astiffened joint, is sufficient to prevent or ameliorate the stiffenedjoint or one or more of its symptoms. Ameliorating the stiffened jointincludes slowing the course of the progression of the joint stiffness orreducing the severity of later-developing joint stiffness.

As used herein, a “subject” is an animal, such as a mammal, including aprimate (e.g., a human), a non-human primate, (e.g., a monkey and achimpanzee), a non-primate (e.g., a cow, a pig, a camel, a llama, ahorse, a goat, a rabbit, a sheep, a hamster, a guinea pig, a cat, a dog,a rat, a mouse, a horse, and a whale), or a bird (e.g., a duck or agoose). In an embodiment, the subject is a human, such as a human beingassessed for a stiffened joint, a human at risk for developing astiffened joint, a human having a stiffened joint, and/or a human beingtreated for a stiffened joint.

As used herein, the terms “treat,” “treatment,” “treating,” or“amelioration” refer to therapeutic treatments, wherein the object is toreverse, alleviate, ameliorate, inhibit, slow down or stop theprogression or severity of a condition, e.g., a condition associatedwith fibrosis, e.g. a fibrotic disease and disorder. The term “treating”includes reducing or alleviating at least one adverse effect or symptomof a fibrotic disease and disorder (e.g., inflammation, stiffening of ajoint, contracture of a joint, contracture of a joint not caused bymuscle contracture, contracture of a joint associated with musclecontracture, pain, loss of mobility, difficulty breathing, musclestiffness, muscle dysfunction, skin dimpling, keloid scarring,burn-associated scarring). Treatment is generally “effective” if one ormore symptoms or clinical markers are reduced. Alternatively, treatmentis “effective” if the progression of a disease is reduced or halted.That is, “treatment” includes not just the improvement of symptoms ormarkers, but also a cessation of, or at least slowing of, progress orworsening of symptoms compared to what would be expected in the absenceof treatment. Beneficial or desired clinical results include, but arenot limited to, alleviation of one or more symptom(s), diminishment ofextent of disease, stabilized (i.e., not worsening) state of disease,delay or slowing of disease progression, amelioration or palliation ofthe disease state, remission (whether partial or total), and/ordecreased mortality, whether detectable or undetectable. The term“treatment” of a disease also includes providing relief from thesymptoms or side-effects of the disease (including palliativetreatment).

As used herein, “prevention” or “preventing,” when used in reference toa stiffened joint, refers to a reduction in the likelihood that asubject, e.g., a human subject, will develop a symptom associated withsuch a stiffened joint, or a reduction in the frequency and/or durationof a symptom associated with a stiffened joint. The likelihood ofdeveloping a stiffened joint is reduced, for example, when a subjecthaving one or more risk factors for a stiffened joint either fails todevelop a stiffened joint or develops a stiffened joint with lessseverity relative to a population having the same risk factors and notreceiving treatment as described herein. The failure to develop astiffened joint, or the reduction in the development of a symptomassociated with stiffened joint (e.g., by at least about 10%), or theexhibition of delayed symptoms (e.g., delayed by days, weeks, months oryears) is considered effective prevention.

As used herein, the term “administering,” refers to the placement of atherapeutic (e.g., an microcapsule comprising an antifibrotic agentdescribed herein) or composition as disclosed herein into a subject by amethod or route which results in at least partial delivery of the agentto the subject. Compositions, e.g., pharmaceutical compositioncomprising agents as disclosed herein can be administered by anyappropriate route which results in an effective treatment in thesubject.

As used herein, a “subject” means a human or animal. Usually the animalis a vertebrate such as a primate, rodent, domestic animal or gameanimal. Primates include, for example, chimpanzees, cynomolgus monkeys,spider monkeys, and macaques, e.g., Rhesus. Rodents include, forexample, mice, rats, woodchucks, ferrets, rabbits and hamsters. Domesticand game animals include, for example, cows, horses, pigs, deer, bison,buffalo, feline species, e.g., domestic cat, canine species, e.g., dog,fox, wolf, avian species, e.g., chicken, emu, ostrich, and fish, e.g.,trout, catfish and salmon. In some embodiments, the subject is a mammal,e.g., a primate, e.g., a human. The terms, “individual,” “patient” and“subject” are used interchangeably herein.

Preferably, the subject is a mammal. The mammal can be a human,non-human primate, mouse, rat, dog, cat, horse, or cow, but is notlimited to these examples. Mammals other than humans can beadvantageously used as subjects that represent animal models of diseasee.g., a disease or disorder associated with fibrosis. A subject can bemale or female.

A subject can be one who has been previously diagnosed with oridentified as suffering from or having a disease or disorder in need oftreatment (e.g., a disease or disorder associated with fibrosis) or oneor more complications related to such a disease or disorder, andoptionally, have already undergone treatment for the disease or disorderor the one or more complications related to the disease or disorder.Alternatively, a subject can also be one who has not been previouslydiagnosed as having such disease or disorder (e.g., a disease ordisorder associated with fibrosis) or related complications. Forexample, a subject can be one who exhibits one or more risk factors forthe disease or disorder or one or more complications related to thedisease or disorder or a subject who does not exhibit risk factors.

The term “microparticle” includes but is not limited to microsphere,microgranules, microsponges, or any non-spherical particles within thespecified dimensions with an internal matrix capable of encapsulation ofthe agent e.g., relaxin.

The term “microparticle” refers to particles with a diameter that ispreferably less than 500um, and more preferably between 1 um and 50 um.Microparticles may also include nanoparticles. Nanoparticles refer to aparticle having a diameter that is preferably less than 1um and greaterthan 10 nm.

As used herein, an “agent” refers to e.g., a molecule, protein, peptide,antibody, or nucleic acid, that inhibits expression of a polypeptide orpolynucleotide, or binds to, partially or totally blocks stimulation,decreases, prevents, delays activation, inactivates, desensitizes, ordown regulates the activity of the polypeptide or the polynucleotide.Agents that inhibit SerpinB1, e.g., inhibit expression, e.g.,translation, post-translational processing, stability, degradation, ornuclear or cytoplasmic localization of a polypeptide, or transcription,post transcriptional processing, stability or degradation of apolynucleotide or bind to, partially or totally block stimulation, DNAbinding, transcription factor activity or enzymatic activity, decrease,prevent, delay activation, inactivate, desensitize, or down regulate theactivity of a polypeptide or polynucleotide. An agent can act directlyor indirectly.

The term “agent” as used herein means any compound or substance such as,but not limited to, a small molecule, nucleic acid, polypeptide,peptide, drug, ion, etc. An “agent” can be any chemical, entity ormoiety, including without limitation synthetic and naturally-occurringproteinaceous and non-proteinaceous entities. In some embodiments, anagent is nucleic acid, nucleic acid analogues, proteins, antibodies,peptides, aptamers, oligomer of nucleic acids, amino acids, orcarbohydrates including without limitation proteins, oligonucleotides,ribozymes, DNAzymes, glycoproteins, siRNAs, lipoproteins, aptamers, andmodifications and combinations thereof etc. In certain embodiments,agents are small molecule having a chemical moiety. For example,chemical moieties included unsubstituted or substituted alkyl, aromatic,or heterocyclyl moieties including macrolides, leptomycins and relatednatural products or analogues thereof. Compounds can be known to have adesired activity and/or property, or can be selected from a library ofdiverse compounds.

The agent can be a molecule from one or more chemical classes, e.g.,organic molecules, which may include organometallic molecules, inorganicmolecules, genetic sequences, etc. Agents may also be fusion proteinsfrom one or more proteins, chimeric proteins (for example domainswitching or homologous recombination of functionally significantregions of related or different molecules), synthetic proteins or otherprotein variations including substitutions, deletions, insertion andother variants.

Methods and compositions described herein comprise an antifibroticagent, e.g., relaxin or functional variant thereof. As used herein,“Relaxin 2” refers to the gene encodes a member of the relaxin subfamilyand insulin superfamily of peptide hormones. Sequences for relaxin 2,also known as H2; RLXH2; H2-RLX; bA12D24.1.1; and bA12D24.1.2, are knownfor a number of species, e.g., human relaxin 2 (NCBI Gene ID: 6019)polypeptide (e.g., NCBI Ref Seq NP_001316120.1) and mRNA (e.g., NCBI RefSeq NM_001329191.2). Relaxin 2 can refer to human Relaxin 2, includingnaturally occurring variants, molecules, and alleles thereof. Relaxin 2refers to the mammalian Relaxin 2 of, e.g., mouse, rat, rabbit, dog,cat, cow, horse, pig, and the like.

The term “decrease”, “reduced”, “reduction”, or “inhibit” are all usedherein to mean a decrease by a statistically significant amount. In someembodiments, “decrease”, “reduced”, “reduction”, or “inhibit” typicallymeans a decrease by at least 10% as compared to an appropriate control(e.g. the absence of a given treatment) and can include, for example, adecrease by at least about 10%, at least about 20%, at least about 25%,at least about 30%, at least about 35%, at least about 40%, at leastabout 45%, at least about 50%, at least about 55%, at least about 60%,at least about 65%, at least about 70%, at least about 75%, at leastabout 80%, at least about 85%, at least about 90%, at least about 95%,at least about 98%, at least about 99%, or more. As used herein,“reduction” or “inhibition” does not encompass a complete inhibition orreduction as compared to a reference level. “Complete inhibition” is a100% inhibition as compared to an appropriate control.

The terms “increase”, “enhance”, or “activate” are all used herein tomean an increase by a reproducible statistically significant amount. Insome embodiments, the terms “increase”, “enhance”, or “activate” canmean an increase of at least 10% as compared to a reference level, forexample an increase of at least about 20%, or at least about 30%, or atleast about 40%, or at least about 50%, or at least about 60%, or atleast about 70%, or at least about 80%, or at least about 90% or up toand including a 100% increase or any increase between 10-100% ascompared to a reference level, or at least about a 2-fold, or at leastabout a 3-fold, or at least about a 4-fold, or at least about a 5-foldor at least about a 10-fold increase, a 20 fold increase, a 30 foldincrease, a 40 fold increase, a 50 fold increase, a 6 fold increase, a75 fold increase, a 100 fold increase, etc. or any increase between2-fold and 10-fold or greater as compared to an appropriate control. Inthe context of a marker, an “increase” is a reproducible statisticallysignificant increase in such level.

As used herein, a “reference level” refers to a normal, otherwiseunaffected cell population or tissue (e.g., a biological sample obtainedfrom a healthy subject, or a biological sample obtained from the subjectat a prior time point, e.g., a biological sample obtained from a patientprior to being diagnosed with a fibrotic disease or disorder, or abiological sample that has not been contacted with an agent disclosedherein).

As used herein, an “appropriate control” refers to an untreated,otherwise identical cell or population (e.g., a patient who was notadministered an agent described herein, or was administered by only asubset of agents described herein, as compared to a non-control cell).

The term “statistically significant” or “significantly” refers tostatistical significance and generally means a two standard deviation(2SD) or greater difference.

As used herein the term “comprising” or “comprises” is used in referenceto compositions, methods, and respective component(s) thereof, that areessential to the method or composition, yet open to the inclusion ofunspecified elements, whether essential or not.

As used herein the term “consisting essentially of” refers to thoseelements required for a given embodiment. The term permits the presenceof additional elements that do not materially affect the basic and novelor functional characteristic(s) of that embodiment of the invention.

As used herein the term “aliphatic polyester” refers to the followingwithout limitation, poly(lactide), poly(glycolide),poly(lactide-co-glycolide), poly(y-valerolactone), polyethylene glycol(PEG), alginate, agarose, poly(hydroxyvalerate), poly(hydroxybutyrate),poly(3-hydroxybutyrate-co-4-hydroxybutyrate), poly(hydroxyhexanoate),poly(butylene succinate), poly(alkylene alkanoate), polypropylenesuccinate), poly(ethylene succinate), poly(ε-caprolactone),poly(ethylene glycol dimethacrylate), gelatin, collagen, agarose,pectin, poly(lysine), bolaamphiphiles, glycosyl-nucleosides, andfluorocarbon chains.

As used herein the term “vinyl polymer” refers to molecules includingwithout limitation, poly(vinyl alcohols) poly(vinyl chlorides),poly(ethylene), poly(propylene), poly(styrene), poly(styrene sulfonate),poly(vinyl chloride), poly(vinyl alcohol), poly(vinyl acetate),poly(vinyl cyanide), poly(vinyl fluoride), poly(vinyl nitrate),poly(vinyl toluene), poly(vinylpyrrolidone), poly(vinylpolypyrrolidone),pluronic polyol, polyoxamer, poly(uronic acid), poly(anhydride),polyNIPAM, poly(acrylates, poly(acrylamides), poly(betaines), tween (20,40, 60, 80), decyl glucoside glycerol monostearate, glycerolmonolaurate, sorbitan monolaurate, sorbitan monostearate, triton x-100,carboxylmethylcellulose, hypromellose, and pluronic F-127. In someembodiments, the listed molecules may be utilized for theiremulsification and stabilization properties.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows microparticles are prepared with batch-to-batch consistencyand low polydispersity. (top) PLGA microparticles encapsulatingrelaxin-2 show spherical morphology. (bottom) They are synthesized withlow polydispersity and a hydrodynamic radius of 7.6 µm as indicated bydynamic light scattering analysis.

FIG. 2 shows relaxin-2 maintains its fold and stability duringencapsulation and release at physiological temperature. (left) Circulardichroism spectroscopy indicates that relaxin-2 is unaffected by thedouble emulsion process and is able to maintain its secondary alphahelical structure during PLGA microparticle synthesis. (right) Relaxin-2is stable at 37° C. for up to 2 months without any loss of alpha helicalstructure. 1-4 h spectra are all overlaid.

FIG. 3 shows encapsulation within PLGA microparticles allows for thesustained release of relaxin-2. (top) Over the course of 30 days, therelaxin-2-loaded PLGA microparticles release 60 % of the initialrelaxin-2. The release is slow in the first three weeks and increasesrapidly after three weeks. (bottom) The microparticles degrade overtime. Beginning after two weeks, the smooth surface of the particles islost as hydrolosys occurs on the polymeric surface. After 4 weeks, theparticles become porous, which account for the increase in release rateof relaxin-2. Scale bar = 2 µm.

FIG. 4 shows relaxin-2 is released in vitro for up to four weeks and isable to decrease collagen expression. (top) Over the course of fourweeks, the relaxin-2 was released at an effective dose as noted by thesignificant downregulation in collagen I expression compared to theuntreated control in synoviocytes. (bottom) Densitometry reveals thatcollagen I is reduced to approximately 50 % after the

FIG. 5 shows large relaxin-2-loaded microparticles are not endocytosedby murine macrophages in vitro. (left) Fluorescently-labelled controlPLGA-microparticles (red) with average diameter of 4.5 µm areendocytosed by RAW 264.7 murine macrophages after 24 h of incubation.(right) Fluorescently-labelled Relaxin-2 loaded microparticles areexcluded from RAW 264.7 macrophages and do not fluoresce internally.Images on top are captured at 10X magnification, and images on bottomare captured at 20X magnification.

FIG. 6 shows structures of supramolecular gel constituents have lowmolecular weight and regions with various physical properties. The bolabis urea molecule 1 and glucosyl-nucleoside fluorinate amphiphile bothhave regions of hydrophobicity (red), aromaticity (blue), andhydrophilicity (black). These regions favor a combination ofhydrophobic, pi-pi stacking, and hydrogen bonding with each otherrespectively

FIG. 7 shows Relaxin-2 can be successfully loaded into an amphiphilichydrogel without compromising its structure. Relaxin-2 is tolerant toincubation for at least one hour at 95° C. as indicated by circulardichroism, which shows its alpha-helical conformation unperturbed(left). Relaxin-2-loaded amphiphiles will gelate with relaxin-2incorporated inside of them (right).

FIG. 8 shows rheological properties of bola bis urea 1. (left) Gel 1 at1, 2, and 5 wt % densities all exhibit solid-like materials up to afrequency of 100 rad/s as indicated by their storage (G′) and loss (G″)moduli. In all gels, G′ > G″, where G′/G″ remains approximately constantand independent of wt %. The absolute values of G′ and G″ arelogarithmically proportional to the gel density. (right) The linearviscoelastic regions were determined from a strain sweep ranging from0.01 to 100 % strain. The 5 % gel showed exhibited the smallest LVERwhere G″ > G′ at 2.2 % strain. The 2 % gel and 1 % gels transition fromthe LVER at 5.8 and 6.4 % strain, respectively (vertical, dotted lines)

FIG. 9 shows rheological properties of glycosyl-nucleoside florinatedamphiphile 2. (left) Gel 2 at 1, 1.5, and 5 wt % densities all exhibitsolid-like materials up to a frequency of 100 rad/s as indicated bytheir storage (G′) and loss (G″) moduli. The absolute values of G′ andG″ are logarithmically proportional to the gel density. (right) Thelinear viscoelastic regions were determined from a strain sweep rangingfrom 0.01 to 100 % strain. The 5 % gel showed exhibited the smallestLVER where G″ > G′ at 9 % strain. The 2 % gel and 1 % gels transitionfrom the LVER at 20 and 30 % strain, respectively (vertical, dottedlines).

FIG. 10 shows Relaxin-2 is release from both amphiphilic gelformulations with different kinetics over 8 days. Gels made fromamphiphile 1 (green) release relaxin-2 in a significantly slower mannerthan they do for gels made from amphiphile 2 (blue). Almost allrelaxin-2 is released from the gels made from compound 2 over thistimeframe whereas gels made from 1 release approximately 5-fold slower.There is a proportionality observed with the release rate and the wt%for the gels made from 1 whereas this proportionality is absent in gelsmade from 2.

FIG. 11 shows Relaxin-2 therapies improve internal ROM back to baselinelevels, but do not affect external ROM in 7 weeks. (left) The internalROM after suture removal shows constancy in the healthy, non-surgicalgroup (blue). All surgical groups show a decreased post-surgicalbaseline in ROM, however, the mIA and RMP groups show a significantincrease back to healthy baseline by week 2. The vehicle control showssignificantly less ROM than the treatment groups until week 7. (middle)The external ROM of the surgical groups are significantly less and donot improve back to healthy baseline over the course of this study.(right) Total ROM shows a slight improvement of the treatment groupscompared to the vehicle control, however, baseline ROM is not achievedin the course of the study.

FIG. 12 shows histological hallmarks of arthrofibrosis are observed inthe vehicle control, but are absent in treatment and healthy groups.(left) H&E staining of the humeral head and glenohumeral joint show anincreased deposition of fibroblasts near the joint capsule (arrowhead)in the vehicle control. This deposition is significantly less in theheathly control. There is also fibroblast-infiltration in surroundingthe top of the humerus (arrow) that is absent in the healthy control andtreatment groups. (middle) Safranin-O staining shows that thearchitecture of glycosamino glycans is largely unchanged between allgroups. (right) Sirius red staining of the shoulder joint showssignificant deposition of fibroblasts with directional orientation,indicative of fibrosis. Delineation between the fibrotic capsule andjoint capsule are present except in the vehicle control (arrows).

FIG. 13 shows sustained release of Relaxin-2 improves total ROM.Induction of contracture is consistent between the vehicle (n=9) andloaded depot (n=7) groups (time = 0). Both the internal (middle) andtotal (right) range of motion show recovery at the later time pointsindicating efficacy of relaxin release into the joint space.

DETAILED DESCRIPTION

Shoulder contracture affects approximately 2% of the U.S. population, orapproximately six million individuals. While women are more oftenaffected than men, there is no known genetic or racial predilection(Robinson C.M. et al., J. Bone Joint Surg. Br. 2012, 94(1):1-9; EwaldA., Am. Fam. Physician 2011, 83(4):417-22). Shoulder contracturerecovery is arduous and protracted with a significant number of patientsnever regaining full joint function. The condition affects both qualityof life and productivity. Its predominant feature is painful, gradualloss of both active and passive glenohumeral motion resulting fromprogressive fibrosis of the glenohumeral joint capsule. The contractedcapsule causes pain, especially when it is stretched suddenly, andproduces a mechanical restraint to motion. The disease course of primary(idiopathic) shoulder contracture begins with the slow onset (over 2 to9 months) of pain and stiffness that progressively restricts bothpassive and active range of motion (ROM) in the glenohumeral joint(Sharma S., Annals of the Royal College of Surgeons of England 201193(5):343-4; discussion 5-6). The pain may sharpen at night, leavingpatients unable to sleep on the affected side. Subsequently, the paingenerally abates over a period of 4 to 12 months, but stiffness severelyrestricts ROM, particularly in the external rotational plane. There is aslow improvement in ROM over a period of 2 to 4 years. Secondaryshoulder contracture has a similar presentation and progression butresults from a known intrinsic or extrinsic cause (Sheridan M.A. andHannafin J.A., Orthop. Clin. North Am. 2006, 37(4):531-9). Secondaryshoulder contracture following trauma or surgery has a 100% incidence tovarying degrees after these events and requires prolonged physicaltherapy, with original motion not always restored.

Shoulder contracture pathology is a thickened glenohumeral joint capsulewith adhesions obliterating the axillary fold. The fibrotic capsuleadheres to itself and the anatomic neck of the humerus, intraarticularvolume is diminished, and synovial fluid in the joint is significantlydecreased (Hand G.C. et al., J. Bone Joint Surg. Br. 2007,89(7):928-32). Biopsy of the capsule shows a chronic inflammatoryinfiltrate, an absence of synovial lining, and subsynovial fibrosis(Ozaki J. et al., J. Bone Joint Surg. Am. 1989, 71(10): 1511-5; WileyA.M., Arthroscopy 1991, 7(2):138-43; Rodeo S.A. et al., J. Orthop. Res.1997, 15(3):427-36). Patient biopsy samples confirm the presence ofT-cells, B-cells, synovial cells, fibroblasts and transformingmyofibroblasts, along with type-I and type-III collagen (Rodeo S.A. etal., J. Orthop. Res. 1997, 15(3):427-36; Bunker T.D. et al., J. BoneJoint Surg. Br. 2000, 82(5):768-73). Gene and protein expression assayshave found products related to fibrosis, inflammation, andchondrogenesis (Hagiwara Y. et al., Osteoarthritis Cartilage 2012,20(3):241-9), including increased COL1A1 and COL1A3, interleukin-6,platelet-derived growth factor (PDGF), fibroblast growth factors (FGF)and inhibitors of the matrix metalloproteinases (TIMPs), as well asdecreased activity of matrix metalloproteinases (MMPs). These dataindicate that inflammatory changes initiate the recruitment offibroblasts and immune cells, precipitating the fibrotic process andinappropriate deposition of collagen. Alternatively, fibrotic changesmay occur first, followed by inflammation. In this case, fibrosis mayresult from an underlying disease process, in which cell signalingpathways governing collagen remodeling may be defective (Bunker T.D. etal., J. Bone Joint Surg. Br. 2000, 82(5):768-73). For example, patientstreated with marimastat, a synthetic TIMP, developed shouldercontractures, and when the marimastat was stopped, the disease regressed(Hutchinson J.W. et al., J. Bone Joint Surg. Br. 1998, 80(5):9078).

Shoulder contracture is considered a self-limiting disease, but recoveryis protracted and arduous, with a significant number of patients neverregaining full ROM. The reported outcomes of conservative therapy (i.e.,physical therapy) vary considerably and are highly dependent on how theyare measured (Neviaser A.S. and Neviaser R.J., J. Am. Acad. Orthop.Surg. 2011, 19(9):536-42). Results tend to be more favorable withsubjective outcome measures than with objective outcome measures. In onestudy, 90% of patients treated with minimal therapy reportedsatisfaction with their shoulder function (Griggs S.M. et al., J. BoneJoint Surg. Am. 2000, 82-A(10): 1398-407). However, another that usedobjective outcomes reported residual pain in 50% of patients and motiondeficit in 60% (Shaffer B. et al., J. Bone Joint Surg. Am.1992;74(5):738-46). Mild to moderate symptoms can persist after 4.4years following symptom onset of shoulder contracture. For thoseexperiencing severe disease, such functional impairment interferes withdaily activities and work-related responsibilities (Hand C. et al.,Journal of Shoulder and Elbow Surgery 2008, 17(2):231-6). When patientsdo not respond to conservative management, other treatment options areavailable. Operative intervention in the form of manipulation underanesthesia may restore motion and decrease pain, but it has beenassociated with complications such as fracture, tendon rupture, andneurologic injury (Castellarin G. et al., Archives of Physical Medicineand Rehabilitation 2004, 85(8):1236-40; Hsu S.Y. and Chan K.M.,International Orthopaedics, 1991, 15(2):79-83; Parker R.D. et al.,Orthopedics, 1989, 12(7):989-90). There are reports that manipulation orcapsular release do not offer reliable and consistent results (ShafferB. et al., J. Bone Joint Surg. Am. 1992, 74(5):738-46; Ryans I. et al.,Rheumatology 2005, 44(4):529-35). Accordingly, a more effective andconsistent therapy for joint stiffness is needed

Aspects described herein provide a microparticle for delivering anantifibrotic agent. For example, a microparticle comprising an aliphaticpolyester and an antibribriotic agent. In certain embodiments, themicroparticle further comprises a vinyl polymer.

Another aspect of the invention provides a composition or formulationthat comprises, consists of, or consists essentially of any of themicroparticles described herein.

Other aspects of the invention provide a method for treating a subjecthaving a disease or disorder associated with fibrosis, the methodcomprising administering to said subject in need thereof any of themicroparticles described herein, any of the compositions describedherein, or any of formulations described herein to the subject.

Antifibrotic Agents

Described herein are microparticles that comprise an antifibrotic agent

In one embodiment, the antifibrotic agent is an agonist of the receptorRXFP1. In one embodiment, the antifibrotic agent is human relaxin-2 oran analog or variant.

The term “relaxin” as used herein, refers to a polypeptide belonging tothe relaxin family (e.g., relaxin-2), a relaxin analog (e.g., apolypeptide that binds to a relaxin receptor), or a fragment (e.g., abioactive fragment) or variant of any of the foregoing and/or any agentthat is an agonist of an agent that binds the relaxin receptor family ofproteins (RXFP1, RXFP2, RXFP3, RXFP4).

Relaxin is an approximately 6-kDa protein belonging to the insulinsuperfamily (Sherwood O.D., Endocr. Rev. 2004, 25(2):205-34). Likeinsulin, relaxin is processed from a prepro-form to the mature hormone,containing A and B peptide chains connected by two interchain disulfidebridges and one intrachain disulfide within the A chain (Chan L.J. etal., Protein Pept. Lett. 2011, 18(3):220-9). Relaxin readily decreasescollagen secretion and increases collagen degradation by increasing theexpression of MMPs and decreasing the expression of TIMPs (Samuel C.S.et al., Cell Mol. Life Sci. 2007, 64(12):1539-57). This hormone isinvolved in reproduction, where it inhibits uterine contraction andinduces growth and softening of the cervix to assist child delivery(Parry L.J. et al., Adv. Exp. Med. Biol. 2007, 612:34-48). Recently, ahighly purified recombinant form of H2 relaxin, or human relaxin-2, hasbeen tested in a number of in vitro and in vivo systems to evaluate bothits ability to modify connective tissue and its potential antifibroticproperties. Several studies report that relaxin-2 acts at multiplelevels to inhibit fibrogenesis and collagen overexpression associatedwith fibrosis and is able to prevent and treat pulmonary, renal,cardiac, and hepatic fibrosis (Bennett R.G., Transl. Res. 2009,154(1):1-6). Relaxin treatment of human fibroblasts caused a reductionin levels of collagen types I and III and fibronectin (Unemori E.N. etal., The Journal of Clinical Investigation 1996, 98(12):2739-45). Invivo, relaxin-2 decreased collagen build-up in the lung induced bybleomycin and improved the overall amount of fibrosis (Unemori E.N. etal., The Journal of Clinical Investigation 1996, 98(12):2739-45). Incultured renal fibroblasts, epithelial cells and mesangial cells,relaxin-2 decreased TGF-β-induced fibronectin levels and increasedfibronectin degradation (McDonald G.A. et al., American Journal ofPhysiology Renal Physiology 2003, 285(1):F59-67). Relaxin-2 has beenshown to have a rapid pharmacokinetic profile. Previous clinical trialsinvestigating relaxin-2 as a treatment for scleroderma, acute heartfailure, and for the induction of labor through cervical ripening,utilized continuous infusion of relaxin-2 via either intravenousadminstration or subcutaneous administration through a minipump.Efficacy of relaxin-2 requires activation of a transmembrane relaxinreceptor for downstream signalling. Previous clinical trials utilizedcontinuous infusion in an attempt to overcome pharmacokineticlimitations. The localized, sustained release of relaxin-2 achievessustained receptor activation without necessitating continuousadministration.

Unless specified to the contrary, the term “relaxin” as used hereinencompasses a relaxin or an analog, a fragment (e.g., a bioactivefragment) or a variant thereof. The term “relaxin or an analog, afragment or a variant thereof” encompasses any member of therelaxin-like peptide family which belongs to the insulin superfamily.The relaxin-like peptide family includes relaxin-like (RLN) peptides,e.g., relaxin- 1 (RLN1), relaxin-2 (RLN2) and relaxin-3 (RLN3), and theinsulin-like (INSL) peptides, e.g., INSL3, INSL4, INSL5 and INSL6.Representative sequences of human RLN1 are listed herein as SEQ ID NOS:4-7; representative sequences of human RLN2 are listed herein as SEQ IDNOS: 1-3; representative sequences of human RLN3 are listed herein asSEQ ID NOS: 8-10; a representative sequence of human INSL3 is listedherein as SEQ ID NO: 11; representative sequences of human INSL4 arelisted herein as SEQ ID NOS: 12-13; representative sequences of humanINSL5 are listed herein as SEQ ID NOS. 14-15; and a representativesequence of human INSL6 is listed herein as SEQ ID NO: 16. In someembodiments, the term “relaxin or an analog, a fragment or a variantthereof may encompass any polypeptide having at least 70%, e.g., atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, 96%,97%, 98% or at least 99% sequence identity with any of SEQ ID NOS: 1-16,as well as any polypeptide sequence that comprises any of SEQ ID NOS:1-16. In one embodiment of the invention, the relaxin includes RLN1,RLN2 or RLN3. In one embodiment, the relaxin is relaxin-1. In anotherembodiment, the relaxin is relaxin-3. In a preferred embodiment, therelaxin is relaxin-2. In another embodiment of the invention, therelaxin includes INSL3, INSL4, INSL5 or INSL6. In one embodiment, therelaxin is INSL3. In one embodiment, the relaxin is INSL4. In oneembodiment, the relaxin is INSL5. In one embodiment, the relaxin isINSL6.

In some embodiments, the relaxin is recombinantly produced, for examplein a bacterial, mammalian or yeast host cell. In other aspects therelaxin has been fully or partially chemically synthesized.

In some embodiments, the term relaxin encompasses any natural,synthetic, or semisynthetic composition that is capable of interactingwith a relaxin family protein receptors (RXFP1, RXFP2, RXFP3, RXPF4)that impacts the form, function, or activity of the receptor. Thesecompounds include but are not limited to native relaxin-2, relaxin-2variants, polypeptides, DNA or RNA polynucleotides, small molecules, aswell as any of the previously listed compounds conjugated to, orassociated with, the relaxin-2 protein.

The term “relaxin or an analog, a fragment or a variant thereof” mayalso encompass any member the relaxin-like peptide family includesrelaxin-like (RLN) peptides, e.g., relaxin-1 (RLN1), relaxin-2 (RLN2)and relaxin-3 (RLN3), and the insulin-like (INSL) peptides, e.g., INSL3,INSL4, INSL5 and INSL6. Representative sequences of human RLN1 arelisted herein as SEQ ID NOS: 4-7; representative sequences of human RLN2are listed herein as SEQ ID NOS: 1-3; representative sequences of humanRLN3 are listed herein as SEQ ID NOS: 8-10; representative sequence ofhuman INSL3 is listed herein as SEQ ID NO: 11; representative sequencesof human INSL4 are listed herein as SEQ ID NOS: 12-13; representativesequences of human INSL5 are listed herein as SEQ ID NOS. 14-15; andrepresentative sequence of human INSL6 is listed herein as SEQ ID NO:16. The term “relaxin or an analog, a fragment or a variant thereof”also in some embodiments encompasses any polypeptide having at least70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, atleast 95% or at least 99% sequence identity with any of SEQ ID NOS:1-16, as well as any polypeptide sequence that comprises any of SEQ IDNOS: 1-16. In one embodiment of the formulation, the relaxin includesRLN1, RLN2 or RLN3. In one embodiment, the relaxin is relaxin-2. Inanother embodiment, the relaxin includes INSL3, INSL4, NSL5 or INSL6.

The term “relaxin or an analog, a fragment or a variant thereof” also insome embodiments may encompass any mutant member of the relaxin-likepeptide family. Such mutant may be, e.g., a RLN1, RLN2, RLN3, INSL3,INSL4, INSL5 or INSL6 comprising one or more mutations, e.g.,substitutions, additions or deletions of one or more amino acids (nativeor non-native) in the known sequence of RLN1, RLN2, RLN3, INSL3, INSL4,INSL5 or INSL6. For example, a mutant member of the relaxin-like peptidefamily may comprise any naturally occurring or artificially producedvariants of RLN1, RLN2, RLN3, INSL3, INSL4, INSL5 or INSL6.

The term “relaxin fragment” or “a fragment of relaxin” as used hereinencompasses a fragment of a relaxin, i.e., a partial sequence of anymember of the relaxin-like peptide family, that retains its ability totreat stiffened joints through interaction with the relaxin familyreceptors. Examples include those sequences described in European PatentOffice Application No. EP1641824B1 (Relaxin superfamily peptideanalogues), the entire contents of which are incorporated herein byreference.

The term “relaxin analog” or an “analog of relaxin” includes anynon-relaxin polypeptide sequence that possesses the biological activityof relaxin, i.e., the ability to interact with the relaxin familyreceptors. In one embodiment, such polypeptide sequence may compriseprolactin or an analog, a fragment or a variant thereof. In anotherembodiment, such sequence may comprise the truncated B-chain analogue ofrelaxin known as B7-33, described in ACS Appl. Mater. Interfaces 2019,11, 49, 45511-45519.

In some embodiments, the term agent or “relaxin analog” also includes arelaxin receptor agonist, e.g., any agent, such as a small molecule, apolypeptide, a polynucleotide or a polysaccharide, that can bind to andactivate a relaxin receptor, e.g., one or more of RXFP1, RXFP2, RXFP3and RXFP4. For example, a relaxin receptor agonist may be a polypeptidecomprising the receptor binding site of relaxin. A relaxin receptoragonist may also be a polypeptide comprising any other sequence capableof binding to and activating the relaxin receptor, e.g., RXFP1, RXFP2,RXFP3 and RXFP4. Other examples include those agonists described in U.S.Pat. Application No. US20130237481A1 (Modified relaxin polypeptides andtheir uses), U.S. Pat. Application No. US20180222960A1 (Modified relaxinpolypeptides comprising a pharmacokinetic enhancer and uses thereof),U.S. Pat. Application No. US8445635B2 (Modified H2 relaxin for tumorsuppression), European Patent Office Application No. EP3067365A1 (Humanrelaxin analogue, pharmaceutical composition of same, and pharmaceuticalapplication of same), and U.S. Pat. Application No. US20180222960A1(Modified relaxin polypeptides comprising a pharmacokinetic enhancer anduses thereof) the entire contents of which are incorporated herein byreference.

The term “relaxin or an analog, a fragment or a variant thereof”includes any recombinantly produced relaxin, such as, e.g., Serelaxin(RLX030) developed by Novartis. Methods for producing recombinantrelaxin, e.g., relaxin-2, are described, .e.g., in U.S. Patent No.5,464,756, the entire contents of which are incorporated herein byreference. The recombinantly produced relaxin or analog, fragment orvariant thereof may comprise a relaxin sequence, e.g., RLN1, RLN2, RLN3,INSL3, INSL4, INSL5 or INSL6, and a histidine (His) tag to aid in thepurification of the relaxin after being recombinantly produced.

The relaxin or analog, fragment or variant thereof may also comprise oneor more chemical modifications, e.g., chemical groups covalentlyattached to the relaxin or an analog, a fragment or a variant thereof.Such chemical groups may include, e.g., carbohydrates or other polymers,e.g., polyethylene glycol (PEG), e.g., polypeptide, e.g. one or morelipids (Design and Synthesis of Potent, Long-Acting Lipidated Relaxin-2Analogs, Bioconjugate Chem. 2019, 30, 1, 83-89). Other examples includefragments or variants described in U.S. Pat. Application No.US2018/0326079 (NOVELFATTYACIDS AND THEIR USE IN CONJUGATION TOBIOMOLECULES), US9,931,372B2 (SYNTHETIC APELIN FATTYACID CONJUGATES WITHIMPROVED HALF-LIFE), the entire contents of which are incorporatedherein by reference.

In some embodiments, relaxin or an analog, a fragment or a variantthereof is co-administered with ML290 or its analog, fragment or avariant to prolong or enhance the effects of RXLP1 activation (Kocan,M., et al. Sci. Rep., 2017).

In some embodiments, the term relaxin includes relaxin attached, e.g.,covalently attached, to an immunoglobulin or a fragment of animmunoglobulin, e.g., an antibody or a fragment of an antibody, forexample, the immunoglobulin fusion proteins described in WO 2017/100540.In other embodiments, the term relaxin does not include relaxinattached, e.g., covalently attached, to an immunoglobulin or a fragmentof an immunoglobulin.

Microparticles, and Compositions and Formulations Thereof

Aspects described herein relate to a microparticle comprising analiphatic polyester and an antifibriotic agent. Exemplary aliphaticpolyesters include poly-lactide-co-glycolide, or polycaprolactone.

In one embodiment, the microparticle further comprises a vinyl polymer.Exemplary vinyl polymers include poly(vinyl alcohol) orpoly(pyrrolidone).

Another aspect herein is a microparticle comprising an aliphaticpolyester and an antifibrotic agent, the microparticles have a diameterof 1-100 µm.

Another aspect herein is a microparticle comprising an aliphaticpolyester and an antifibrotic agent, the antifibrotic agent is relaxinand is present in an amount that is 0.01-33% of total mass.

Another aspect herein is a microparticle comprising an aliphaticpolyester and an antifibrotic agent, the aliphatic polyester is ofmolecular weight 10,000-200,000 daltons.

Another aspect herein is a microparticle comprising an aliphaticpolyester, a vinyl polymer and an antifibrotic agent.

Another aspect herein is a microparticle comprising an aliphaticpolyester, a vinyl polymer and an antifibrotic agent, the microparticleshave a diameter of 1-100 µm.

Another aspect herein is a microparticle comprising an aliphaticpolyester, a vinyl polymer and an antifibrotic agent, the antifibroticagent is relaxin and is present in an amount that is 0.01-33% of totalmass.

Another aspect herein is a microparticle comprising an aliphaticpolyester, a vinyl polymer and an antifibrotic agent, the aliphaticpolyester is of molecular weight 10,000-200,000 daltons.

Another aspect herein is a PLGA microparticle comprising an aliphaticpolyester and an antifibrotic agent, the microparticles have a diameterof 1-100µm.

Another aspect herein is a PLGA microparticle comprising an aliphaticpolyester and an antifibrotic agent, the antifibrotic agent is relaxinand is present in an amount that is 0.01-33% of total mass.

Another aspect herein is a PLGA microparticle comprising an aliphaticpolyester and an antifibrotic agent, the aliphatic polyester is ofmolecular weight 10,000-200,000 daltons.

Another aspect herein is a PLGA microparticle comprising an aliphaticpolyester, a vinyl polymer and an antifibrotic agent, the microparticleshave a diameter of 1-50 µm.

Another aspect herein is a PLGA microparticle comprising an aliphaticpolyester, a vinyl polymer and an antifibrotic agent, the antifibroticagent is relaxin and is present in an amount that is 0.1-33% of totalmass.

Another aspect herein is a PLGA microparticle comprising an aliphaticpolyester, a vinyl polymer and an antifibrotic agent, the aliphaticpolyester is of molecular weight 10,000-200,000 daltons.

In one embodiment, where a feature of a formulation (such as anantifibrotic agent, a relaxin, a vinyl polymer, an aliphatic polyester,etc) is in a specified amount expressed in a mass percentage;percent-by-weight; percent of total mass or the like; unless indicatedotherwise, the percentage is based on microparticles that are notsuspended in a solution. In such situations where a claimed formulationincludes microparticles suspended in a solution, the mass percentagestill refers to the mass percentage in the microparticles, and not thetotal solution that includes the microparticles and the suspensionsolution.

Pharmaceutical Compositions

Various aspects herein relate to a composition comprising any of themicroparticles described herein. In various embodiments, the compositionis a pharmaceutical composition.

As used herein, the term “pharmaceutical composition” can include anymaterial or substance that, when combined with an active ingredient(e.g., an antifibrotic agent, such as relaxin), allows the ingredient toretain biological activity and is non-reactive with the subject’s immunesystem. Examples include, but are not limited to, any of the standardpharmaceutical carriers such as a phosphate buffered saline solution,emulsions such as oil/water emulsion, and various types of wettingagents. The phrase “pharmaceutically acceptable” is employed herein torefer to those compounds, materials, compositions, and/or dosage formswhich are, within the scope of sound medical judgment, suitable for usein contact with the tissues of human beings and animals withoutexcessive toxicity, irritation, allergic response, or other problem orcomplication, commensurate with a reasonable benefit/risk ratio.

The phrase “pharmaceutically acceptable carrier” as used herein means apharmaceutically acceptable material, composition or vehicle, such as aliquid or solid filler, diluent, excipient, solvent or encapsulatingmaterial, involved in carrying or transporting the subject agents fromone organ, or portion of the body, to another organ, or portion of thebody. The term “pharmaceutically acceptable carrier” excludes tissueculture media. Each carrier must be “acceptable” in the sense of beingcompatible with the other ingredients of the formulation, for examplethe carrier does not decrease the impact of the agent on the treatment.In other words, a carrier is pharmaceutically inert. The terms“physiologically tolerable carriers” and “biocompatible deliveryvehicles” are used interchangeably. Non-limiting examples ofpharmaceutical carriers include particle or polymer-based vehicles suchas nanoparticles, microparticles, polymer microspheres, or polymer-drugconjugates.

In some embodiments, the pharmaceutical composition is a liquid dosageform or solid dosage form. Liquid dosage forms for oral administrationinclude, but are not limited to, pharmaceutically acceptable emulsions,microemulsions, solutions, suspensions, syrups and elixirs. In addition,the liquid dosage forms can contain inert diluents commonly used in theart such as, for example, water or other solvents, solubilizing agentsand emulsifiers such as ethyl alcohol, isopropyl alcohol, ethylcarbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propyleneglycol, 1,3-butylene glycol, dimethylformamide, oils (in particular,cottonseed, groundnut, com, germ, olive, castor, and sesame oils),glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fattyacid esters of sorbitan, and mixtures thereof. Besides inert diluents,the oral compositions can also include adjuvants such as wetting agents,emulsifying and suspending agents, sweetening, flavoring, and perfumingagents.

In some embodiments, the liquid dosage form is prepared at or near thepoint of care by reconstituting or resuspending a provided lyophilisateor lyophilized powder of a formulation disclosed herein using a diluentsolution.

Solid dosage forms for oral administration include capsules, tablets,pills, powders, and granules. In such solid dosage forms, the agentsdescribed herein are mixed with at least one inert, pharmaceuticallyacceptable excipient or carrier such as sodium citrate or dicalciumphosphate and/or a) fillers or extenders such as starches, lactose,sucrose, glucose, mannitol, and silicic acid, b) binders such as, forexample, carboxymethylcellulose, alginates, gelatin,polyvinylpyrrolidinone, sucrose, and acacia, c) humectants such asglycerol, d) disintegrating agents such as agar-agar, calcium carbonate,potato or tapioca starch, alginic acid, certain silicates, and sodiumcarbonate, e) solution retarding agents such as paraffin, f) absorptionaccelerators such as quaternary ammonium compounds, g) wetting agentssuch as, for example, cetyl alcohol and glycerol monostearate, h)absorbents such as kaolin and bentonite clay, and i) lubricants such astalc, calcium stearate, magnesium stearate, solid polyethylene glycols,sodium lauryl sulfate, and mixtures thereof. In the case of capsules,tablets and pills, the dosage form can also comprise buffering agents.

Solid compositions of a similar type can also be employed as fillers insoft and hard-filled gelatin capsules using such excipients as lactoseor milk sugar as well as high molecular weight polyethylene glycols, andthe like. The solid dosage forms of tablets, dragées, capsules, pills,and granules can be prepared with coatings and shells such as entericcoatings and other coatings well known in the pharmaceutical formulatingart. They can optionally contain opacifying agents and can also be of acomposition that they release the active ingredient(s) only, orpreferentially, in a certain part of the intestinal tract, optionally,in a delayed manner. Examples of embedding compositions that can be usedinclude polymeric substances and waxes. Solid compositions of a similartype can also be employed as fillers in soft and hard-filled gelatincapsules using such excipients as lactose or milk sugar as well as highmolecular weight polyethylene glycols, and the like.

In some emodiments, the solid dosage form is a lyophilized powder.

In some dosage forms, the lyophilized powder solid dosage form isintended to be resuspended or reconstituted with diluent.

The agent can also be in micro-encapsulated form with one or moreexcipients as noted above. The solid dosage forms of tablets, dragées,capsules, pills, and granules can be prepared with coatings and shellssuch as enteric coatings, release controlling coatings and othercoatings well known in the pharmaceutical formulating art. In such soliddosage forms, the agent can be admixed with at least one inert diluentsuch as sucrose, lactose and starch. Such dosage forms can alsocomprise, as in normal practice, additional substances other than inertdiluents, e.g., tableting lubricants and other tableting aids such asmagnesium stearate and microcrystalline cellulose. In the case ofcapsules, tablets and pills, the dosage forms can also comprisebuffering agents. They can optionally contain opacifying agents and canalso be of a composition that they release the active ingredient(s)only, or preferentially, in a certain part of the intestinal tract,optionally, in a delayed manner. Examples of embedding compositionswhich can be used include polymeric substances and waxes.

Pharmaceutical compositions include formulations suitable for oraladministration may be provided as discrete units, such as tablets,capsules, cachets, syrups, elixirs, prepared food items, microemulsions,solutions, suspensions, lozenges, or gel-coated ampules, each containinga predetermined amount of the active compound; as powders or granules;as solutions or suspensions in aqueous or non-aqueous liquids; or asoil-in-water or water-in-oil emulsions.

Accordingly, formulations suitable for rectal administration includegels, creams, lotions, aqueous or oily suspensions, dispersible powdersor granules, emulsions, dissolvable solid materials, douches, and thelike can be used. The formulations are preferably provided as unit-dosesuppositories comprising the active ingredient in one or more solidcarriers forming the suppository base, for example, cocoa butter.Suitable carriers for such formulations include petroleum jelly,lanolin, polyethyleneglycols, alcohols, and combinations thereof.Alternatively, colonic washes with the rapid recolonization deploymentagent of the present invention can be formulated for colonic or rectaladministration.

The present invention provides sustained release formulations fordelivering a polypeptide therapeutic to a subject in need thereof. Thesustained release formulations of the invention consist of a hydrogel,microparticle or some matrix encapsulation of the agent. One example ofthe agent is relaxin. The sustained release comprises the agent e.g.,relaxin encapsulated by or chemically bound to the depot supportmaterial via a linker. The linker may, comprise a polymer, anon-cleavable linker, or a cleavable linker, either through chemical orenzymatic means. The depot may be formed in situ following mixing of theagent with the material. The depot may be formed prior to mixing of therelaxin with the material.

The sustained release formulation comprising the agent e.g., relaxin maybe in the form of a hydrogel or microparticle which comprises one ormore polymers. The polymers that may be used in a sustained releaserelaxin formulation may include, without limitation, polyethylene glycol(PEG), alginate, agarose, poly(ethylene glycol dimethacrylate),polylactic acid, polyglycolic acid, poly-lactide-co-glycolide, gelatin,collagen, agarose, pectin, poly(lysine), polyhydroxybutyrate,poly-epsilon-caprolactone, polyphosphazines, poly(vinyl alcohol),poly(alkylene oxide), poly(ethylene oxide), poly(allylamine),poly(acrylate), poly(4-aminomethylstyrene), pluronic polyol, polyoxamer,poly(uronic acid), poly(anhydride), poly(vinylpyrrolidone),bolaamphiphiles, glycosyl-nucleosides, and fluorocarbon chains.

In some embodiments of any of the aspects described herein, an agent isadministered to a subject by controlled- or delayed-release means.Ideally, the use of an optimally designed controlled-release preparationin medical treatment is characterized by a minimum of drug substancebeing employed to cure or control the condition in a minimum amount oftime. Advantages of controlled-release formulations include: 1) extendedactivity of the drug; 2) reduced dosage frequency; 3) increased patientcompliance; 4) usage of less total drug; 5) reduction in local orsystemic side effects; 6) minimization of drug accumulation; 7)reduction in blood level fluctuations; 8) improvement in efficacy oftreatment; 9) reduction of potentiation or loss of drug activity; and10) improvement in speed of control of diseases or conditions. (Kim,Cherng-ju, Controlled Release Dosage Form Design, 2 (TechnomicPublishing, Lancaster, Pa.: 2000)). Controlled-release formulations canbe used to control a compound of formula (I)′s onset of action, durationof action, plasma levels within the therapeutic window, and peak bloodlevels. In particular, controlled- or extended-release dosage forms orformulations can be used to ensure that the maximum effectiveness of anagent is achieved while minimizing potential adverse effects and safetyconcerns, which can occur both from under-dosing a drug (i.e., goingbelow the minimum therapeutic levels) as well as exceeding the toxicitylevel for the drug.

A variety of known controlled- or extended-release dosage forms,formulations, and devices can be adapted for use with any agentdescribed herein. Examples include, but are not limited to, thosedescribed in U.S. Pat. Nos.: 3,845,770; 3,916,899; 3,536,809; 3,598,123;4,008,719; 5674,533; 5,059,595; 5,591,767; 5,120,548; 5,073,543;5,639,476; 5,354,556; 5,733,566; and 6,365,185, each of which isincorporated herein by reference in their entireties. These dosage formscan be used to provide slow or controlled-release of one or more activeingredients using, for example, hydroxypropylmethyl cellulose, otherpolymer matrices, gels, permeable membranes, osmotic systems (such asOROS® (Alza Corporation, Mountain View, Calif. USA)), multilayercoatings, microparticles, liposomes, or microspheres or a combinationthereof to provide the desired release profile in varying proportions.Additionally, ion exchange materials can be used to prepare immobilized,adsorbed salt forms of the disclosed compounds and thus effectcontrolled delivery of the drug. Examples of specific anion exchangersinclude, but are not limited to, DUOLITE® A568 and DUOLITE® AP143(Rohm&Haas, Spring House, Pa. USA).

In some embodiments, any aforementioned polymers, prior to or afterloading of relaxin, may be characterized (e.g. size, molecular weight,charge, secondary structure, and purity) by techniques including, butnot limited to, gel permeation chromatography, high performance liquidchromatography, ultra-performance liquid chromatography, MALDI-TOF massspectroscopy, viscometry, and light scattering (e.g. multi-angle, lowangle laser).

In some embodiments, the rate of release of relaxin may be characterizedby techniques including, but not limited to, high performance liquidchromatography, ultra-performance liquid chromatography, fast proteinliquid chromatography, enzyme linked immunosorbent assay, and ligandbinding assay. In some embodiments, the release rate of relaxin ismeasured as the concentration of relaxin in any biologically relevantliquid solution or suspension or medium (e.g. saline, mammalian cellculture media, synthetic synovial fluid, synovial fluid, serum,synthetic serum, plasma, synthetic plasma and deionized water) that theformulation is also in. In specific embodiments, the formulation andbiologically relevant liquid solution or suspension is maintained at aspecific temperature. In specific embodiments, the formulation andbiologically relevant liquid solution or suspension is agitated or mixedat a set or varying rate of motion. In specific embodiments, theconcentration of relaxin released into the biologically relevant liquidsolution or suspension is measured using a direct enzyme linkedimmunosorbent assay. In specific embodiments, the concentration ofrelaxin released into the biologically relevant liquid solution orsuspension is measured using an indirect enzyme linked immunosorbentassay. In specific embodiments, the concentration of relaxin releasedinto the biologically relevant liquid solution or suspension is measuredusing a sandwich enzyme linked immunosorbent assay. In a preferredembodiment, the concentration of relaxin released into the biologicallyrelevant liquid solution or suspension is measured using the HumanRelaxin-2 Quantikine ELISA Kit from Bio-techne corporation.

In some embodiments, the size and morphology (e.g. diameter, sphericity,and porosity) of relaxin microparticles may be characterized bytechniques including, but not limited to, dynamic light scattering,coulter counter, microscopy, sieve analysis, dynamic image analysis,static image analysis, and laser diffraction.

In some embodiments, the total loaded content of relaxin in relaxinmicroparticles (e.g. percent of relaxin as weight/volume, percent ofrelaxin as weight/weight) may be characterized by techniques including,but not limited to, mass balance, limited to, high performance liquidchromatography, ultra-performance liquid chromatography, fast proteinliquid chromatography, enzyme linked immunosorbent assay, and ligandbinding assay. In some embodiments, the formulation may be purified anddissolved to assess total loaded content of relaxin.

In some embodiments, the total loaded content (i.e. mass) of relaxin inrelaxin microparticles is measured as the concentration of relaxin inany liquid solution, suspension or medium (e.g. saline, mammalian cellculture media, synthetic synovial fluid, synovial fluid, serum,synthetic serum, plasma, synthetic plasma, methylene chloride,acetonitrile, ethyl acetate, and deionized water) that the totalformulation may be dissolved in. In specific embodiments, theconcentration of relaxin after formulation dissolution in the liquidsolution, suspension, or medium is measured using a direct enzyme linkedimmunosorbent assay. In specific embodiments, the concentration ofrelaxin after formulation dissolution in the liquid solution,suspension, of medium is measured using an indirect enzyme linkedimmunosorbent assay. In specific embodiments, the concentration ofrelaxin after formulation dissolution in the liquid solution,suspension, of medium is measured using a sandwich enzyme linkedimmunosorbent assay. In a preferred embodiment, the concentration ofrelaxin after formulation dissolution in the liquid solution,suspension, of medium is measured using the Human Relaxin-2 QuantikineELISA Kit from Bio-techne corporation.

In certain embodiments, the sustained release formulation comprises ofPEG, e.g., a linear PEG or a branched PEG. In certain embodiments, themolecular weight of the PEG is more than 0.2 kDa, more than 0.5 kDa,more than 1 kDa, more than 5 kDa, more than 10 kDa, or more than 20 kDa

In some embodiments, the hydrogel comprises of PEG-based crosslinkerswith an internal thioester that will be reacted with dendrons to preparehydrogels. These hydrogels may be prepared in varying weight percent tomodulate mechanical properties. In specific embodiments the internalthioester allows for controlled dissolution through the use of acysteine methyl ester solution. In specific embodiments, the gelsmaterial properties including, but not limited to, release profile,young’s modulus, sheer modulus, hydrophobicity, and, elasticity can bevaried through modification of the thioester moiety to modulate materialproperties of hydrogel.

In one embodiment of any aspect herein, the aliphatic polyester ispoly-lactide-co-glycolide.

In one embodiment of any aspect herein, the aliphatic polyester ispolycaprolactone.

In one embodiment of any aspect herein, the aliphatic polyester is ofmolecular weight 10,000-200,000 daltons; 10,000-150,000 daltons; or25,000-125,000 daltons; or 40,00-100,000 daltons; 10,000-30,000 daltons;30,000-50,000 daltons; 50,000-70,000 daltons; 70,000-90,000 daltons;90,000-120,000 daltons; or 120,000-150,000 daltons.

In one embodiment of any aspect herein, the aliphatic polyester isterminated by an ester functional group.

In one embodiment of any aspect herein, the aliphatic polyester isterminated by an alkyl-ester functional group.

In one embodiment of any aspect herein, the aliphatic polyester isterminated by a carboxylic acid functional group.

In one embodiment of any aspect herein, the aliphatic polyester isterminated by an amine functional group, an isocyanate functional group,an isothiocyanate functional group, a benzoyl fluoride functional group,a maleimide functional group, an iodoacetamide functional group, a2-thiopyridine functional groups, a 3-arylpropiolonitrile functionalgroup, a diazonium salt, an aldehyde, a ketone, an azide, an alkyne, acyclooctyne, or a phosphine. In one embodiment of any aspect herein,these functional groups that allow for bioconjuntion between the PLGAand a biomolecule.

In one embodiment of any aspect herein, the formulation comprises avinyl polymer that is poly(vinyl alcohol).

In one embodiment of any aspect herein, the formulation comprises avinyl polymer that is poly(pyrrolidone).

In one embodiment of any aspect herein, the formulation comprises avinyl polymer that is of molecular weight 10,000-200,000 daltons;10,000-150,000 daltons; or 25,000-125,000 daltons; or 40,00-100,000daltons; 10,000-30,000 daltons; 30,000-50,000 daltons; 50,000-70,000daltons; 70,000-90,000 daltons; 90,000-120,000 daltons; or120,000-150,000 daltons.

In one embodiment of any aspect herein, the diameter of themicroparticles is 1-100 µm.

In one embodiment of any aspect herein, the diameter of themicroparticles is 1-75 µm; or 1-50 µm; or 5-50 µm; or 25-50 µm; or 30-50µm; or 40-50 µm; or 5-10 µm; 5-8 µm; 8-12 µm; 12-18 µm; 18-25 µm; 25-35µm; 35-45 µm; 45-50 µm; 1 µm; 2 µm; 3 µm; 4 µm; 5 µm; 6 µm; 7 µm; 8 µm;9 µm; 10 µm; 15 µm; 20 µm; 25 µm; 30 µm; 35 µm; 40 µm; 45 µm; 50 µm; 75µm; 100 µm; 150 µm; or 200 µm.

In one embodiment of any aspect herein, the aliphatic polyester ispoly-lactide-co-glycolide with a molar ratio of 15:85 - 25:75,lactide:glycolide; poly-lactide-co-glycolide with a molar ratio of25:75 - 35:65, lactide:glycolide; poly-lactide-co-glycolide with a molarratio of 35:65 - 45:55, lactide:glycolide; poly-lactide-co-glycolidewith a molar ratio of 45:55 - 55:45, lactide:glycolide;poly-lactide-co-glycolide with a molar ratio of 55:45 - 65:35,lactide:glycolide; poly-lactide-co-glycolide with a molar ratio of65:35 - 75:25, lactide:glycolide; poly-lactide-co-glycolide with a molarratio of 75:25 - 85:15, lactide:glycolide; poly-lactide-co-glycolidewith a molar ratio of about 50:50, lactide:glycolide;poly-lactide-co-glycolide with a molar ratio of about 45:55,lactide:glycolide; poly-lactide-co-glycolide with a molar ratio of about55:45, lactide:glycolide; poly-lactide-co-glycolide with a molar ratioof about 40:60, lactide:glycolide; or poly-lactide-co-glycolide with amolar ratio of about 60:40, lactide:glycolide.

In one embodiment of any aspect herein, the formulation comprises avinyl polymer that is about 0.01-0.1% of total mass; 0.1-0.3% of totalmass; 0.2-0.9% of total mass; 0.3-0.7% of total mass; 0.4-0.6% of totalmass; 0.3-0.6% of total mass; 0.6-1.0% of total mass; 1.0-5.0% of totalmass; 5.0-10.0% of total mass; 10.0-30.0% of total mass; 0.1% of totalmass; 0.2% of total mass; 0.3% of total mass; 0.4% of total mass; 0.5%of total mass; 0.6% of total mass; 0.7% of total mass; 0.8% of totalmass; 0.9% of total mass; 10% of total mass; 15% of total mass; 20% oftotal mass; 25% of total mass; 30% of total mass; or 33% of total mass.

In one embodiment of any aspect herein, the antifibrotic agent is0.005-5% of the total formulation mass. In one embodiment of any aspectherein, the antifibrotic agent is 0.01-10%, 0.01-33%, or 0.1-5% of thetotal formulation mass; or 0.2-4% of the total formulation mass; or0.3-3% of the total formulation mass; or 0.5-2% of the total formulationmass; or 0.5-1.5% of the total formulation mass; or 0.5-3% of the totalformulation mass; or 1-2% of the total formulation mass; or 1-5% of thetotal formulation mass; or 3-7% of the total formulation mass; or 5-10%of the total formulation mass.

In one embodiment of any aspect herein, the antifibrotic agent is about0.005-0.01% of the total formulation mass; 0.01-0.05% of the totalformulation mass; 0.05-0.1% of the total formulation mass; 0.1-0.5% ofthe total formulation mass; 0.5-1.0% of the total formulation mass;1.0-2.5% of the total formulation mass; 2.5-5.0% of the totalformulation mass; 0.25% of the total formulation mass; 0.5% of the totalformulation mass; 0.75% of the total formulation mass; 1% of the totalformulation mass; 1.25% of the total formulation mass; 1.5% of the totalformulation mass; 1.75% of the total formulation mass; 2% of the totalformulation mass; 2.5% of the total formulation mass; 3% of the totalformulation mass; or 5% of the total formulation mass.

In one embodiment of any aspect herein, the formulation comprises PLGAmicroparticles with a PLGA molar ratio that is about 50:50lactide:glycolide, a relaxin loaded at about 1% by weight of themicroparticles, and PVA in a concentration of about 0.5% by weight.

In one embodiment of any aspect herein, the formulation comprises PLGAmicroparticles with a PLGA molar ratio that is about 50:50lactide:glycolide, a relaxin loaded at about 1% by weight of themicroparticles and PVA in a concentration of about 0.0% by weight

In one embodiment of any aspect herein, the formulation comprises PLGAmicroparticles with a PLGA molar ratio that is about 60:40lactide:glycolide, a relaxin loaded at about 1% by weight of themicroparticles, and PVA in a concentration of about 0.5% by weight.

In one embodiment of any aspect herein, the formulation comprises PLGAmicroparticles with a PLGA molar ratio that is 40:60 lactide:glycolide,a relaxin loaded at about 1% by weight of the microparticles, and PVA ina concentration of about 0.5% by weight.

In one embodiment of any aspect herein; the formulation comprisesmicroparticles suspended in a liquid solution.

In one embodiment of any aspect herein; the formulation comprisesmicroparticles suspended in a sodium chloride liquid solution.

In one embodiment of any aspect herein; the formulation comprisesmicroparticles suspended in a sodium chloride liquid solution; thesodium chloride is 0.5-1.5 w/w%; or between 0.75-1.25 w/w%; or about 0.5w/w%; or about 0.6 w/w%; or about 0.7 w/w%; or about 0.8 w/w%; or about0.9 w/w%; or about 1.0 w/w%; or about 1.1 w/w%; or about 1.2 w/w%; orabout 1.3 w/w%; or about 1.4 w/w%; or about 1.5 w/w% of the liquidsolution.

In one embodiment of any aspect herein; the formulation comprisesmicroparticles suspended in a sodium carboxymethylcellulose solution.

In one embodiment of any aspect herein, the formulation comprisesmicroparticles suspended in a sodium carboxymethylcellulose solution;the sodium carboxymethylcellulose solution is 0.1-1.0 w/w%; or between0.25-0.75 w/w%; or about 0.1 w/w%; or about 0.2 w/w%; or about 0.3 w/w%;or about 0.4 w/w%; or about 0.5 w/w%; or about 0.6 w/w%; or about 0.7w/w%; or about 0.8 w/w%; or about 0.9 w/w%; or about 1.0 w/w% of theliquid solution.

In one embodiment of any aspect herein, the formulation is a sustainedrelease formulation.

In one embodiment of any aspect herein, the formulation is a sustainedrelease formulation the antifibrotic agent is released over an extendedperiod of time.

For relaxin to have a sustained clinical antifibrotic effect, it isphysiologically desirable for the temporal concentration of relaxin tobe above the minimum effective concentration for a sustained duration. Abolus dose of relaxin is reported to not be effective in animals. Asustained dose of relaxin is reported to be effective in animals.

A constant sustained dose of relaxin may be achieved by the relase ofrelaxin from a microparticle with a linear rate of release (i.e. onehaving no bolus effect or burst-release effect).

In one embodiment of any aspect herein, the formulation is a sustainedrelease formulation the antifibrotic agent is released over an extendedperiod of least 1 day; or at least 2 days; or at least 3 days; or atleast 4 days; or at least 5 days; or at least 6 days; or at least 1week; or at least 2 weeks; or at least 3 weeks; or at least 4 weeks; orat least 5 weeks, or at least 6 weeks; or at least 8 weeks; or at least9 weeks; at least 10 weeks; or at least 12 weeks; or at least 15 weeks;or between 1-5 days; or between 2-5 days; or between 1-2 days; orbetween 2-3 days; or between 3-4 days; or between 4-5 days; or between3-10 days; or between 1-15 weeks; or between 2-10 weeks; or between 4-8weeks; or between 8-15 weeks; or about 1 day; or about 2 days; or about3 days; or about 4 days; or about 5 days; or about 6 days; or about 1week; or about 2 weeks; or about 3 weeks; or about 4 weeks; or about 5weeks; or about 6 weeks; or about 7 weeks; or about 8 weeks; or about 9weeks; or about 10 weeks, or more.

Treatment of Diseases or Disorders Associated With Fibrosis

In some embodiments, a formulation as described herein is administeredto a subject. In some embodiments, a formulation as described herein isused to treat an organ or location on the body of a subject, a diseaseor indication in a subject and or using an administration route asdescribed in Table 1 and/or Table 2.

TABLE 1 Administrati on routes and targets Sites (non-joints) Lung,kidney, liver, heart, skin, eye; tendons, osteotendinous junctions,tendon-bone interfaces, entheses, or muscle-tendon insertions,mentioning a slew of tendons throughout the body Sites (joints) Jaw,spine, shoulder, elbow, wrist, hand, finger, hip, knee, ankle, foot,toe; or any other synovial or non-synovial joint Routes ofadministration JOINT INJECTIONS (JI): Intraarticular, periarticular,intracapsular, pericapsular NON-JOINT DENSE CONNECTIVE TISSUE INJECTIONS(NJDCTI): intraligamentous, periligamentous, intratendinous,peritendinous, intraosteotendinous, or periosteotendinous;intramusculotendinous, perimusculotendinous, perimuscularly,OTHER,NON-ORTHOPEDIC: intravenously, intramuscularly, subcutaneously,intradermally, intranasally, orally, transcutaneously(ionto/electrophoresis), mucosally, gel, cream, ointment, lotion, drop,suppository, spray, liquid, powder, pulmonary inhalation, ocular.Indications When to administer treatment During or just after a medicalprocedure; for patients with stiffened joint or at risk for stiffenedjoint (treatment or prophylactic)

TABLE 2 General causes of fibrosis Idiopathic, injury (trauma, medicalprocedure e.g. surgery), immobility for whatever reason, inflammation,or disease / medical condition Diseases/conditions: joints (admin viajoint injection) adhesive capsulitis (injury, idiopathic, post-surgical,post-implant) Diseases/conditions: lung (admin via inhalation)idiopathic pulmonary fibrosis, cystic fibrosis, hypertensionDiseases/conditions: liver hepatitis B or C, long-term alcohol abuse,non-alcoholic steatohepatitis, non-alcoholic fatty liver disease,Cholestasis, autoimmune hepatitis cirrhosis Diseases/conditions: kidneychronic kidney disease, end-stage renal disease, renal interstitialfibrosis Diseases/conditions: heart heart failure, myocardialinfarction, aortic stenosis, hypertrophic cardiomyopathyDiseases/conditions: intestine Crohn’s disease, inflammatory boweldisease, enteropathies Diseases/conditions: skin (admin via intradermalinjection, or transdermal) scleroderma, keloids, hypertrophic scars,cellulite Diseases/conditions: urogenital / gynecological Peyronie’sdisease, uterine fibroids, urethral strictures Diseases/conditions:ocular Congenital Fibrosis of the Extraocular Muscles, subretinalfibrosis, epiretinal fibrosis, corneal fibrosis Diseases/conditions:connective tissue, fascia Dupuytren’s disease, capsular contracture ofbreast, Plantar fibromatosis, Diseases/conditions: neuromuscular (adminvia joint or peri-joint injection) Duchenne, Becker, congenital, andother muscular dystrophies, SMA, Charcot-Marie-Tooth, arthrogryposis,ALS, club foot, post-polio, CP.

In some embodiments, a method is provided in which the method involvesidentifying a subject diagnosed with one or more diseases selected fromthe group of diseases listed in Table 1 or Table 2 and administering aformulation of the invention to the subject. In some embodiments, amethod is provided in which the method involves identifying a subjectdiagnosed with one or more diseases selected from the group of diseasesconsisting of Duchenne Muscular Dystrophy, Becker Muscular Dystrophy,Spinal Muscular Atrophy-Type I, Spinal Muscular Atrophy-Type II, SpinalMuscular Atrophy-Type III, Spinal Muscular Atrophy-Type IV, CerebralPalsy, Stroke, Traumatic Brain Injury, peripheral nerve injury, andArthrogryposis Multiplex Congenita, fibrosis of the humeroradial joint,fibrosis of the humeroulnar joint, fibrosis of the glenohumeral joint,fibrosis of the tibiofemoral joint, fibrosis of the acetabulofemoraljoint, fibrosis of the talocrural joint, fibrosis of thetemporomandibular joint, fibrosis of the metacarpophalangeal joint,fibrosis of the metatarsophalangeal joint, fibrosis of theperi-articular musculature and cellulite and administering to saidpatient a composition or formulation of the invention.

Stiffened Joint

Various compositions and methods disclosed herein may be useful fortreating various aspects, precursors and related disorders of jointstiffness. Joint stiffness is a significant public health issue withcurrent treatment options providing varied and limited outcomes. Jointstiffness can affect any joint in the body, such as a shoulder joint, anelbow joint, a wrist joint, a finger joint, a hip joint, a knee joint,an ankle joint, a toe joint, the spine and the jaw A shoulder joint isoften affected by joint stiffness, which is also termed a shouldercontracture, and is also known as “frozen shoulder”.

Shoulder contracture affects approximately 2% of the U.S. population, orapproximately six million individuals. While women are more oftenaffected than men, there is no known genetic or racial predilection(Robinson C.M. et al., J. Bone Joint Surg. Br. 2012, 94(1): 1-9; EwaldA., Am. Fam. Physician 2011, 83(4):417-22). Shoulder contracturerecovery is arduous and protracted with a significant number of patientsnever regaining full joint function. The condition affects both qualityof life and productivity. Its predominant feature is painful, gradualloss of both active and passive glenohumeral motion resulting fromprogressive fibrosis of the glenohumeral joint capsule. The contractedcapsule causes pain, especially when it is stretched suddenly, andproduces a mechanical restraint to motion. The disease course of primary(idiopathic) shoulder contracture begins with the slow onset (over 2 to9 months) of pain and stiffness that progressively restricts bothpassive and active range of motion (ROM) in the glenohumeral joint(Sharma S., Annals of the Royal College of Surgeons of England 201193(5):343-4; discussion 5-6). The pain may sharpen at night, leavingpatients unable to sleep on the affected side. Subsequently, the paingenerally abates over a period of 4 to 12 months, but stiffness severelyrestricts ROM, particularly in the external rotational plane. There is aslow improvement in ROM over a period of 2 to 4 years. Secondaryshoulder contracture has a similar presentation and progression butresults from a known intrinsic or extrinsic cause (Sheridan M.A. andHannafin J.A., Orthop. Clin. North Am. 2006, 37(4):531-9). Secondaryshoulder contracture following trauma or surgery has a 100% incidence tovarying degrees after these events and requires prolonged physicaltherapy, with original motion not always restored.

Shoulder contracture pathology is a thickened glenohumeral joint capsulewith adhesions obliterating the axillary fold. The fibrotic capsuleadheres to itself and the anatomic neck of the humerus, intraarticularvolume is diminished, and synovial fluid in the joint is significantlydecreased (Hand G.C. et al., J. Bone Joint Surg. Br. 2007,89(7):928-32). Biopsy of the capsule shows a chronic inflammatoryinfiltrate, an absence of synovial lining, and subsynovial fibrosis(Ozaki J. et al., J. Bone Joint Surg. Am. 1989, 71(10): 1511-5; WileyA.M., Arthroscopy 1991, 7(2): 138-43; Rodeo S.A. et al., J. Orthop. Res.1997, 15(3):427-36). Patient biopsy samples confirm the presence ofT-cells, B-cells, synovial cells, fibroblasts and transformingmyofibroblasts, along with type-I and type-III collagen (Rodeo S.A. etal., J. Orthop. Res. 1997, 15(3):427-36; Bunker T.D. et al., J. BoneJoint Surg. Br. 2000, 82(5):768-73). Gene and protein expression assayshave found products related to fibrosis, inflammation, andchondrogenesis (Hagiwara Y. et al., Osteoarthritis Cartilage 2012,20(3):241-9), including increased COL1A1 and COL1A3, interleukin-6,platelet-derived growth factor (PDGF), fibroblast growth factors (FGF)and inhibitors of the matrix metalloproteinases (TIMPs), as well asdecreased activity of matrix metalloproteinases (MMPs). These dataindicate that inflammatory changes initiate the recruitment offibroblasts and immune cells, precipitating the fibrotic process andinappropriate deposition of collagen. Alternatively, fibrotic changesmay occur first, followed by inflammation. In this case, fibrosis mayresult from an underlying disease process, in which cell signalingpathways governing collagen remodeling may be defective (Bunker T.D. etal., J. Bone Joint Surg. Br. 2000, 82(5):768-73). For example, patientstreated with marimastat, a synthetic TIMP, developed shouldercontractures, and when the marimastat was stopped, the disease regressed(Hutchinson J.W. et al., J. Bone Joint Surg. Br. 1998, 80(5):907-8).

Shoulder contracture is considered a self-limiting disease, but recoveryis protracted and arduous, with a significant number of patients neverregaining full ROM. The reported outcomes of conservative therapy (i.e.,physical therapy) vary considerably and are highly dependent on how theyare measured (Neviaser A.S. and Neviaser R.J., J. Am. Acad. Orthop.Surg. 2011, 19(9):536-42). Results tend to be more favorable withsubjective outcome measures than with objective outcome measures. In onestudy, 90% of patients treated with minimal therapy reportedsatisfaction with their shoulder function (Griggs S.M. et al., J. BoneJoint Surg. Am. 2000, 82-A(10): 1398-407). However, another that usedobjective outcomes reported residual pain in 50% of patients and motiondeficit in 60% (Shaffer B. et al., J. Bone Joint Surg. Am.1992;74(5):738-46). Mild to moderate symptoms can persist after 4.4years following symptom onset of shoulder contracture. For thoseexperiencing severe disease, such functional impairment interferes withdaily activities and work-related responsibilities (Hand C. et al.,Journal of Shoulder and Elbow Surgery 2008, 17(2):231-6). When patientsdo not respond to conservative management, other treatment options areavailable. Operative intervention in the form of manipulation underanesthesia may restore motion and decrease pain, but it has beenassociated with complications such as fracture, tendon rupture, andneurologic injury (Castellarin G. et al., Archives of Physical Medicineand Rehabilitation 2004, 85(8):1236-40; Hsu S.Y. and Chan K.M.,International Orthopaedics, 1991, 15(2):79-83; Parker R.D. et al.,Orthopedics, 1989, 12(7):989-90). There are reports that manipulation orcapsular release do not offer reliable and consistent results (ShafferB. et al., J. Bone Joint Surg. Am. 1992, 74(5):738-46; Ryans I. et al.,Rheumatology 2005, 44(4):529-35). Accordingly, a more effective andconsistent therapy for joint stiffness is needed.

Encapsulation of biologically active agents into biocompatible andbiodegradable polymeric matrices prior to administration prolongseffective therapeutic levels in a patient. Hydrogels and microparticlesare implantable structures. They are desirable for therapeutic deliverydue to designs that are biocompatible, made of non-toxic constituents,not immunogenic or cause irritation and do not hinder the target tissuestructurally or mechanically. Significantly, they can be administeredlocally to the area of interest. One material that is extensively usedfor microencapsulation and prolonged release of small molecule drugs,DNA and proteins is poly(lactic-co-glycolic) acid (PLGA). PLGA isbiocompatible and releases its payload both through diffusion of out ofthe polymer matrix and via breakdown of the polymer matrix. Thebreakdown occurs through hydrolysis of PLGA, catalyzed by the body’saqueous environment, into lactic acid and glycolic acid, which arebyproducts of cellular metabolism. It is considered safe foradministration to humans by the United States Food and DrugAdministration (Han, F.C. et al., Front. Pharmacol. 2016, 7(185)). PLGAmicroparticles can be optimized for sustained drug release by adjustingthe ratio of lactic acid to glycolic acid and the emulsificationprotocol. However, it has been reported to cause a foreign bodyresponse.

Techniques for encapsulation of a biologically active agent insidelactide, glycolide copolymer microparticles are known. The productiontechniques generally include either the use of two solvent phases,stabilizer, and the biologically active agent dissolved or solvated intoone of the phases or the use of water/oil/water (w/o/w) or water/oil(w/o) emulsions. In the first mentioned production technique, the twophases, biologically active agent and stabilizer are emulsifier and thenone of the phases is removed, leaving behind a microparticle withstabilized, loaded agent. In the w/o/w fabrication technique, theinitial water phase contains or does not contain the biologically activecompound, is emulsified within the organic phase containing thedissolved polymeric matrix and then emulsified within the second aqueousphase. The removal of the organic phase leaves behind a microparticlecontaining or not containing the biologically active compound. That saidthe specific methods and PLGA compositions used highly depend on theencapsultant and a general procedure/composition does not exist for allencapsultants.

Another promising material for use specifically in the field ofregenerative medicine and tissue maintenance as a drug delivery systemare hydrogels. These hydrogels are polymeric networks capable ofencapsulating biologically active agents. Hydrogels possess relevantbiological properties such as biocompatibility, sheering thinningcharacteristics, biodegradation, and do not impact the stability oractivity of the loaded biologically active agent.

One such formulation of hydrogels involves a network of low molecularweight gelators (LMWG) that act as injectable scaffolds for biomedicalapplications. They have tunable physiochemical and biological propertiesdue to their supramolecular structure stemming from the self-assembly ofsmall molecules. Specifically, LMWGs with bolaamphiphiles consisting ofa N-thymine glycosylated head groups linked to a lipidic moiety viaeither urea or amide functions have shown to be fast-gelling with highin vivo stability and do not activate macrophages (FIG. 1 ) (Ramen, F.A.et al., Biomaterials. 2017, 145: 72-80). Another LMWG formulationutilizes a combination of glycosyl-nucleosides and fluorocarbon chainsas amphiphiles that self-assemble into highly organized structures thatincreases stability of hydrogel formulations (FIG. 1 ) (Godeau, G., etal., Tetrahedron Letters 2010, 51: 1012-1015). They demonstratednumerous advantageous properties, including biocompatibility, controlover structure and purity, easy handling procedure to allow forincorporation of proteins, mechanical stability and are non-toxic tocells (Godeau, G., et al., Tetrahedron Letters 2010, 51: 1012-1015;Ramen, F.A. et al., Biomaterials. 2017, 145: 72-80).

Another formulation of hydrogels is the use of a PEG-based hydrogel. Inthis formulation the PEG-based hydrogel would include polymeric PEGmatrix with a biologically active agent either linked or encapsulated tothe matrix. Encapsulation would occur through localization of thebiological agent into the hydrogel during polymerization. Release of theagent would occur through diffusion out of the hydrogel into the tissue.In the case of a chemical bond between the agent and the hydrogel, itwould be either a cleavable or non-cleavable connection. If cleavable,the linkage would be either rely upon an enzymatic or non-enyzmaticbased mechanism.

Rigid contracture or fibrosis (arthrofibrosis) of the major articularjoints is a severely limiting comorbidity and sequela of many neuromotordegenerative disorders. It presents as an accumulation of fibroticcollagenous tissue within the joint and manifests as a painful andlongstanding restriction of joint range of motion (ROM), contributing topoor mobility and requiring home care assistance orinstitutionalization.

Stiffened joint may be most limiting in the shoulders, elbows, knees,hips, wrists, and ankles of patients with progressive neuromotordisorders. Degenerative disorders that may be treated by formulationsand methods provided herein and that lead to arthrofibrosis and havedifferent etiologies and include, but are not limited to, Duchenne (DMD)and Becker (BMD) muscular dystrophies, Congenital Muscular Dystrophies(CMD), Spinal Muscular Atrophy (SMA), Charcot-Marie-Tooth disease (CMT),arthrogryposis, Emery Dreifus Muscular Dystrophy (EMD), the family ofslow progressive muscular dystrophies (Limb-girdle (LGMD),Fascioscapulohumeral (FSH), Congenital Myotonic (CMMD)), AmyotrophicLateral Sclerosis (ALS), idiopathic congenital club foot, post-poliosyndrome, all forms of cerebral palsy (1,2), (CP), stroke, traumaticbrain injury, and peripheral nerve injury. Incidence of these conditionsis on the order of 1-10 / 100,000 population for the dystrophies and 2-3/ 1000 births for cerebral palsy (2). The national cost burden ofmanagement of these conditions is significant, with population-widenational costs just for managing three of these diseases estimated to be$1,023 million (ALS), $787 million (DMD), and $448 million (CMMD)(3).The CDC estimates the overall cost of care for the population ofpatients with cerebral palsy born in the year 2000, will exceed 11.5billion (4). These expenses represent medical as well as non-medicalcosts, and account primarily for musculoskeletal care.

The lack of joint mobility caused by arthrofibrosis in patients with aneuromotor degenerative condition or neuromotor trauma, such as stroke,traumatic brain injury, and peripheral nerve injury, contributes tofurther muscle tone loss, muscle fibrosis and scarring, osteoporosis,secondary deformities such as spinal scoliosis and lower extremityequinus posture, and loss of skin integrity. Ultimately, arthrofibrosisresults in inability to ambulate and limits activities of daily living.In the stages of disease when patients may no longer be ambulatory,joint contracture further burdens nursing care, rest positioning,sitting, and hygiene (1).

At present, prolonged physical therapy, forceful passive stretching,serial casting, and bracing are the only non-operative treatmentmodalities available, with or without botulism toxin supplementation todiminish muscle associated contracture (1). Surgical Interventions toimprove mobility of a fibrosed joint include manipulation underanesthesia, tendon and muscle releases, and articular capsular releaseor resection surgeries of the involved joints (5). Manipulation of ajoint under anesthesia can result in periarticular and shaft fractures,when forceful mobilization of the fibrosed joint introduces more stressto the adjacent osteoporotic bone than it can tolerate. Many patientsare also poor candidates given the intubation and ventilation requiredfor the application of a paralyzing anesthetic agent to counter muscleresistance during a manipulation or surgical release. After prolongedperiods of contracture, acute surgical joint release and manipulationmay also result in severe nerve and vascular stretch injuries, withinconsistent results and variable recurrence rates.

The overall health and quality of life for these patients would begreatly enhanced by an alternative non-operative treatment modalityaimed at resolving joint contracture. This invention, in someembodiments, provides a solution and is a non-surgical office-basedintra-articular injection therapy to be used in conjunction withphysical therapy to release contracted joints over a two to eight-weekperiod.

In various aspects and embodiments, the compositions and methodsprovided herein can be of value to a wide range of subjects. Patientswith neuromotor degenerative disease are a highly managed population,requiring a lifetime of intensive and costly medical and non-medicalsupport. The current standard of care is either conservative treatmentor surgical intervention. In contrast, the compositions and methodsprovided herein may in some embodiments provide a therapeutic benefitwith an in-office injection, eliminating surgery, and offering mobilityto an immobile patient, improving their overall health and quality oflife and reducing the intensity of supportive care. Caretakers,physicians, and specialists will be able to restore joint motion withoutperforming surgery on this at-risk patient population. Patients willbenefit from improved motion and require less physical therapy tomaintain joint mobility. They would retain an independent ability tomobilize and perform activities of daily living for longer periods oftime as their condition progresses. They would enjoy overall improvedmusculoskeletal health and psychosocial benefit. For the payor, theoverall health care cost for the management of these conditions woulddecrease as surgical cost per patient would decrease in addition to thehigher likelihood that a patient would be able to remain at home longerand not require institutional care for sequela of poor mobility orinability to perform adequate care and hygiene at home.

The present invention provides methods for treating or preventing astiffened joint in a subject in need thereof. The methods comprise ofadministering to the subject an effective amount of an agent or ligandof the relaxin family receptors, a relaxin-2 variant, relaxin-2chemically conjugated to a targeting agent, including a single-domaincamelid antibody fragment, a peptide sequence, polynucleotide, or asmall molecule, such that the stiffened joint or surrounding tissue areain the subject is treated.

The current methods for treating a stiffened joint include physicaltherapy or surgical procedures, such as manipulations and releases,which do not offer reliable or consistent results (Diercks R.L. et al.,J. Shoulder Elbow Surg. 2004, 13(5):499-502). Physical therapy involvesprolonged manipulation by a physical therapist and surgical proceduresinvolves invasive surgical release by a surgeon, followed again byprolonged therapy. Another current method, the Ponseti method, involvesserial re-casting after stretching, sometimes with surgical release ofcontracted tendons.

The methods of the invention are, in some embodiments, advantageous ascompared to many currently available methods because they can be used toreliably and effectively treat a stiffened joint or tissue area, whilealso using a minimally invasive procedure, e.g., an intraarticularinjection, which may be performed in an outpatient setting or an office.Thus, some methods of the invention constitute a paradigm shift in themanagement of a stiffened joint, e.g., a shoulder joint, that may resultfrom fibrosis. Some methods of the invention involve minimally invasiveprocedures, e.g., an intraarticular or periarticular injection ofrelaxin-2, e.g., relaxin-2 encapsulated in a sustained releaseformulation. The intraarticular injection may be repeated as neededuntil the stiffened joint is successfully treated, e.g., until motion inthe joint is restored and pain during motion is eliminated. Successfultreatment of a stiffened joint when using some methods of the inventionmay be accomplished significantly faster and more effectively than whenusing the currently available methods.

Pathology of a stiffened joint, e.g., a shoulder joint, includes athickened glenohumeral joint capsule with adhesions obliterating theaxillary fold. The fibrotic capsule adheres to itself and the anatomicneck of the humerus, intraarticular volume is diminished, and synovialfluid in the joint is significantly decreased. Biopsy of the capsuleshows a chronic inflammatory infiltrate, with the presence offibroblasts and transforming myofibroblasts, along with type-I andtype-III collagen. Gene and protein expression assays have foundcomponents related to fibrosis, inflammation, and chondrogenesis,including increased COL1A1 and COL1A3, interleukin-6 (IL-6),platelet-derived growth factor (PDGF), fibroblast growth factors (FGF)and TMPs, as well as decreased MMP activity. This evidence points toinflammatory changes initiating the recruitment of fibroblasts andimmune cells, precipitating the fibrotic process and inappropriatedeposition of excess collagen. Alternatively, it is also possible thatfibrosis occurs first, followed by inflammation; fibrosis beingsecondary to defective cell-signaling pathways governing collagenremodeling.

Without wishing to be bound by a specific theory, it is believed thatthe agent e.g., relaxin, when delivered to or near a joint, e.g., via ahydrogel or particle, intraarticular injection, sustained releaseformulation, promotes collagen degradation, thereby altering thehomeostasis of the extracellular matrix (ECM) in the synovium. Thisadministration results in decreased joint stiffness and increased rangeof motion of the joint.

In one embodiment, the antifibotic agent of the invention isadministered as a monotherapy. In one embodiment, the antifibotic agentof the invention is administered with at least one additionaltherapeutic. Exemplary additional therapeutics include, but are notlimited to, an addition anti-fibrotic therapeutic or physical therapy.

In some embodiments, relaxin or an analog, a fragment or a variantthereof is co-administered with other native anti-fibrotic agents suchas IFN-a, IFN-β, srli B, M3, MMP1, MMP8. Additionally, the use of otheranti-fibrotic agents that target receptors other than the relaxinreceptor: TGF-beta inhibitors (Esbriet, pirfenidone), tyrosine kinaseinhibitors (Ofev, nintedanib). PPAR (peroxisome proliferator-activatedreceptors) agonists (Ianifibranor, IVA337), IL-1 inhibitors (Arcalyst,rilonacept), IL-6 inhibitors (Actmera, tocilizumab), B-cell inhibitors(rituximab), T-cell inhibitors (Orencia, abatacept), lysophosphatidicacid inhibitors (SAR100842, Sanofi), Halofunginone, d-penicillamine,colchicine, cyclosporine, TGF beta blockers, p38 MAPK blockers

Methods for Treating a Stiffened Joint

Some aspects of the present invention provide methods for treating orpreventing a stiffened joint. As used herein, the terms “treating”,“treat” or “treatment” refer to a beneficial or desired resultincluding, but not limited to, alleviation or amelioration of one ormore symptoms associated with a stiffened joint (e.g., pain on movementof the joint, loss of motion of the joint or loss of the range of motionof the joint); diminishing the restriction of movement resulting from astiffened joint; stabilization (i.e., not worsening) of the jointstiffness; amelioration or palliation of the restriction of movementresulting from a stiffened joint (e.g., pain on movement of the joint,loss of motion of the joint or loss of the range of motion of the joint)whether detectable or undetectable.

In some embodiments, methods of the present invention result in atreatment of the stiffened joint, such that pain on movement of thejoint is reduced, e.g., by at least about 10%, at least about 15%, atleast about 20%, at least about 25%, at least about 30%, at least about35%, at least about 40%, at least about 45%, at least about 50%, atleast about 55%, at least about 60%, at least about 65%, at least about70%, at least about 75%, at least about 80%, at least about 85%, atleast about 90%, at least about 95%, or more, and is preferably down toa level accepted within the range of normal for an individual who is notaffected by a stiffened joint.

In some embodiments, methods of the present invention result inrestoration of the movement, or a range of the movement, of a jointaffected by joint stiffness. For example, treatment of the stiffenedjoint according to the methods of the invention may result inrestoration of the movement, or a range of movement, of a joint affectedby joint stiffness, to levels that are at least about 10%, at leastabout 15%, at least about 20%, at least about 25%, at least about 30%,at least about 35%, at least about 40%, at least about 45%, at leastabout 50%, at least about 55%, at least about 60%, at least about 65%,at least about 70%, at least about 75%, at least about 80%, at leastabout 85%, at least about 90%, at least about 95%, or 100% of the levelsaccepted within the range of normal for an individual not affected by astiffened joint.

In some embodiments, prevention or treatment of a stiffened joint in asubject provided by the methods of the present invention is accomplishedwithout significant adverse events, without significant damage tocollagenous structures or tissues in the subject, e.g., collagenousstructures or tissues of the joint, such as articular cartilage of thejoint. For example, methods of the present invention provide preventionand treatment of stiffened joint that do not disrupt architecture of thejoint. Damage to collagenous structures in the body, e.g., collagenousstructures of a joint, may be assessed by methods known in the art,e.g., by measuring levels of various markers in the synovial fluid, suchas Cartilage Oligomeric Matrix Protein (COMP), aggrecans, collagen II,proteoglycans, MMPs and inflammatory mediators and cytokines. Imagingtechniques such as MRI can also be used to visualize the joint and thecartilage architecture.

In some embodiments, when the agent (e.g., relaxin) loaded depot isadministered intraarticularly, prevention or treatments of stiffenedjoint by the methods of the present invention is accomplished withoutsignificant adverse events associated with systemic administration ofrelaxin. In a phase III clinical trial for use of relaxin to treatsystemic sclerosis, some of the patients that received a 24-weeksubcutaneous infusion of relaxin had declines in creatine clearance andrenal adverse events; however renal physiology abnormalities areassociated with systemic sclerosis and may have predisposed the affectedpatients to such renal events when combined with relaxin treatment(Khanna, D., et al., Arthritis and Rheumatism 2009, 60(4): 1102-1111).When relaxin is administered intraarticularly by methods of the presentinvention, serum creatine levels, protein levels in the urine, bloodcell count, hemoglobin concentration in the blood and systolic anddiastolic blood pressure will be monitored during and afteradministration for prevention or treatment of a stiffened joint forindication of renal crisis and hypertension.

One aspect provided herein is a method, said method comprisingidentifying a subject diagnosed with one or more diseases selected fromthe group of diseases listed in Table 1 or Table 2 and administering aformulation of any one of the preceding embodiments to the subject.

Another aspect provided herein is a method, said method comprisingidentifying a subject diagnosed with Duchenne Muscular Dystrophy andadministering to said patient a composition or formulation of any of thepreceding embodiments.

Another aspect provided herein is a method, said method comprisingidentifying a subject diagnosed with Becker Muscular Dystrophy andadministering to said patient a composition or formulation of any of thepreceding embodiments.

Another aspect provided herein is a method, said method comprisingidentifying a subject diagnosed with Spinal Muscular Atrophy, Type I,and administering to said patient a composition or formulation of any ofthe preceding embodiments.

Another aspect provided herein is a method, said method comprisingidentifying a subject diagnosed with Spinal Muscular Atrophy, Type II,and administering to said patient a composition or formulation of any ofthe preceding embodiments.

Another aspect provided herein is a method, said method comprisingidentifying a subject diagnosed with Spinal Muscular Atrophy, Type III,and administering to said patient a composition or formulation of any ofthe preceding embodiments.

Another aspect provided herein is a method, said method comprisingidentifying a subject diagnosed with Spinal Muscular Atrophy, Type IV,and administering to said patient a composition or formulation of any ofthe preceding embodiments.

Another aspect provided herein is a method, said method comprisingidentifying a subject diagnosed with Cerebral Palsy and administering tosaid patient a composition or formulation of any of the precedingembodiments.

Another aspect provided herein is a method, said method comprisingidentifying a subject diagnosed with Arthrogryposis Multiplex Congenitaand administering to said patient a composition or formulation of any ofthe preceding embodiments.

Another aspect provided herein is a method, said method comprisingidentifying a subject with fibrosis of the humeroradial joint andadministering to said patient a composition or formulation of any of thepreceding embodiments.

Another aspect provided herein is a method, said method comprisingidentifying a subject with fibrosis of the humeroulnar joint andadministering to said patient a composition or formulation of any of thepreceding embodiments.

Another aspect provided herein is a method, said method comprisingidentifying a subject with fibrosis of the glenohumeral joint andadministering to said patient a composition or formulation of any of thepreceding embodiments.

Another aspect provided herein is a method, said method comprisingidentifying a subject with fibrosis of the tibiofemoral joint andadministering to said patient a composition or formulation of any of thepreceding embodiments.

Another aspect provided herein is a method, said method comprisingidentifying a subject with fibrosis of the acetabulofemoral joint andadministering to said patient a composition or formulation of any of thepreceding embodiments.

Another aspect provided herein is a method, said method comprisingidentifying a subject with fibrosis of the talocrural joint andadministering to said patient a composition or formulation of any of thepreceding embodiments.

Another aspect provided herein is a method, said method comprisingidentifying a subject with fibrosis of the temporomandibular joint andadministering to said patient a composition or formulation of any of thepreceding embodiments.

Another aspect provided herein is a method, said method comprisingidentifying a subject with fibrosis of the metacarpophalangeal joint andadministering to said patient a composition or formulation of any of thepreceding embodiments.

Another aspect provided herein is a method, said method comprisingidentifying a subject with fibrosis of the metatarsophalangeal joint andadministering to said patient a composition or formulation of any of thepreceding embodiments.

Another aspect provided herein is a method, said method comprisingidentifying a subject with fibrosis of the peri-articular musculatureand administering to said patient a composition or formulation of any ofthe preceding embodiments.

Another aspect provided herein is a method, said method comprisingidentifying a subject with cellulite and administering to said patient acomposition or formulation of any of the preceding embodiments.

Another aspect provided herein is a method, said method comprisingidentifying a subject with interstitial lung disease and administeringto said patient a composition or formulation of any of the precedingembodiments.

Another aspect provided herein is a method, said method comprisingadministering, to any of the preceding subjects, a composition orformulation of any of the preceding embodiments, via inhalation as anaerosol.

Another aspect provided herein is a method, said method comprisingadministering, to any of the preceding subjects, a composition orformulation of any of the preceding embodiments, via intra-articularinjection.

Another aspect provided herein is a method, said method comprisingadministering, to any of the preceding subjects, a composition orformulation of any of the preceding embodiments, via intramuscularinjection.

Another aspect provided herein is a method, said method comprisingadministering, to any of the preceding subjects, a composition orformulation of any of the preceding embodiments, via intradermalinjection.

Another aspect provided herein is a method, said method comprisingadministering, to any of the preceding subjects, a composition orformulation of any of the preceding embodiments, via subcutaneousinjection.

Another aspect provided herein is a method, said method comprisingadministering, to any of the preceding subjects, a composition orformulation of any of the preceding embodiments, via intracapsularinjection.

Another aspect provided herein is a method, said method comprisingadministering, to any of the preceding subjects, a composition orformulation of any of the preceding embodiments, via pericapsularinjection.

Another aspect provided herein is a method, said method comprisingadministering, to any of the preceding subjects, a composition orformulation of any of the preceding embodiments, via intraligamentousinjection.

Another aspect provided herein is a method, said method comprisingadministering, to any of the preceding subjects, a composition orformulation of any of the preceding embodiments, via periligamentousinjection.

Another aspect provided herein is a method, said method comprisingadministering, to any of the preceding subjects, a composition orformulation of any of the preceding embodiments, via intratendinousinjection.

Another aspect provided herein is a method, said method comprisingadministering, to any of the preceding subjects, a composition orformulation of any of the preceding embodiments, via peritendinousinjection.

Another aspect provided herein is a method, said method comprisingadministering, to any of the preceding subjects, a composition orformulation of any of the preceding embodiments, viaintramusculotendinous injection.

Another aspect provided herein is a method, said method comprisingadministering, to any of the preceding subjects, a composition orformulation of any of the preceding embodiments, viaperimusculotendinous injection.

Another aspect provided herein is a method, said method comprisingadministering, to any of the preceding subjects, a composition orformulation of any of the preceding embodiments, via intraosteotendinousinjection.

Another aspect provided herein is a method, said method comprisingadministering, to any of the preceding subjects, a composition orformulation of any of the preceding embodiments, via periosteotendinousinjection.

Another aspect provided herein is a method, said method comprisingadministering, to a subject diagnosed with Duchene’s muscular dystrophy,a composition or formulation of any of the preceding embodiments, viaintramuscular injection.

Another aspect provided herein is a method, said method comprisingadministering, to a subject diagnosed with Duchene’s muscular dystrophy,a composition or formulation of any of the preceding embodiments, viaintraarticular injection.

Another aspect provided herein is a method, said method comprisingadministering, to a subject diagnosed with Becker’s muscular dystrophy,a composition or formulation of any of the preceding embodiments, viaintramuscular injection.

Another aspect provided herein is a method, said method comprisingadministering, to a subject diagnosed with Becker’s muscular dystrophy,a composition or formulation of any of the preceding embodiments, viaintraarticular injection

Another aspect provided herein is a method, said method comprisingadministering, to a subject diagnosed with Spinal Muscular Dystrophy, acomposition or formulation of any of the preceding embodiments, viaintramuscular injection.

Another aspect provided herein is a method, said method comprisingadministering, to a subject diagnosed with Spinal Muscular Dystrophy, acomposition or formulation of any of the preceding embodiments, viaintraarticular injection.

Another aspect provided herein is a method, said method comprisingadministering, to a subject diagnosed with Arthrogryposis MultiplexCongenita, a composition or formulation of any of the precedingembodiments, via intramuscular injection.

Another aspect provided herein is a method, said method comprisingadministering, to a subject diagnosed with Arthrogryposis MultiplexCongenita, a composition or formulation of any of the precedingembodiments, via intraarticular injection.

Another aspect provided herein is a method, said method comprisingadministering, to a subject diagnosed with Cerebral Palsy, a compositionor formulation of any of the preceding embodiments, via intramuscularinjection.

Another aspect provided herein is a method, said method comprisingadministering, to a subject diagnosed with Stroke, a composition orformulation of any of the preceding embodiments, via intramuscularinjection.

Another aspect provided herein is a method, said method comprisingadministering, to a subject diagnosed with Stroke, a composition orformulation of any of the preceding embodiments, via intraarticularinjection.

Another aspect provided herein is a method, said method comprisingadministering, to a subject diagnosed with Traumatic Brain Injury, acomposition or formulation of any of the preceding embodiments, viaintramuscular injection.

Another aspect provided herein is a method, said method comprisingadministering, to a subject diagnosed with Traumatic Brain Injurt, acomposition or formulation of any of the preceding embodiments, viaintraarticular injection.

Another aspect provided herein is a method, said method comprisingadministering, to a subject diagnosed with Peripheral Nerve Injury, acomposition or formulation of any of the preceding embodiments, viaintramuscular injection.

Another aspect provided herein is a method, said method comprisingadministering, to a subject diagnosed with Peripheral Nerve Injury, acomposition or formulation of any of the preceding embodiments, viaintraarticular injection.

Another aspect provided herein is a method, said method comprisingadministering, to a subject diagnosed with Cerebral Palsy, a compositionor formulation of any of the preceding embodiments, via intraarticularinjection.

Another aspect provided herein is a method, said method comprisingadministering any of the preceding embodiments with sizes between 1um-10 µm via inhalation as an aerosol.

Another aspect provided herein is a method, said method comprisingadministering any of the preceding embodiments with sizes between 20um-100 µm via intramuscular injection.

Another aspect provided herein is a method, said method comprisingadministering any of the preceding embodiments with sizes between 5um-50 µum via intraarticular injection.

Another aspect provided herein is a method, said method comprisingadministering, to a subject diagnosed with interstitial lung disease anyof the preceding embodiments via inhalation as an aerosol.

Another aspect provided herein is a method, said method comprisingadministering, to a subject diagnosed with interstitial lung disease anyof the preceding embodiments via inhalation as an aerosol.

Another aspect provided herein is a method, said method comprisingadministering, to a subject diagnosed with interstitial lung disease anyof the preceding embodiments, wherein the diameter of the microparticleis 1-10 µm, via inhalation as an aerosol.

Another aspect provided herein is a method, said method comprisingadministering, to a subject diagnosed with Duchene’s Muscular Dystrophyany of the preceding embodiments, wherein the diameter of themicroparticle is 10-30 µm, via intraarticular injection.

Another aspect provided herein is a method, said method comprisingadministering, to a subject diagnosed with Duchene’s Muscular Dystrophyany of the preceding embodiments, wherein the diameter of themicroparticle is 25-50 µm, via intraarticular injection.

Another aspect provided herein is a method, said method comprisingadministering, to a subject diagnosed with Duchene’s Muscular Dystrophyany of the preceding embodiments, wherein the diameter of themicroparticle is 10-30 µm, via intramuscular injection.

Another aspect provided herein is a method, said method comprisingadministering, to a subject diagnosed with Duchene’s Muscular Dystrophyany of the preceding embodiments, wherein the diameter of themicroparticle is 25-50 µm, via intramuscular injection.

Another aspect provided herein is a method, said method comprisingadministering, to a subject diagnosed with Spinal Muscular Atrophy anyof the preceding embodiments, wherein the diameter of the microparticleis 10-30 µm, via intraarticular injection.

Another aspect provided herein is a method, said method comprisingadministering, to a subject diagnosed with Spinal Muscular Atrophy anyof the preceding embodiments, wherein the diameter of the microparticleis 25-50 µm, via intraarticular injection.

Another aspect provided herein is a method, said method comprisingadministering, to a subject diagnosed with Spinal Muscular Atrophy anyof the preceding embodiments, wherein the diameter of the microparticleis 10-30 µm, via intramuscular injection.

Another aspect provided herein is a method, said method comprisingadministering, to a subject diagnosed with Spinal Muscular Atrophy anyof the preceding embodiments, wherein the diameter of the microparticleis 25-50 µm, via intramuscular injection.

Another aspect provided herein is a method, said method comprisingadministering, to a subject diagnosed with joint arthrofibrosis any ofthe preceding embodiments, wherein the diameter of the microparticle is10-30 µm, via intraarticular injection.

Another aspect provided herein is a method, said method comprisingadministering, to a subject diagnosed with joint arthrofibrosis any ofthe preceding embodiments, wherein the diameter of the microparticle is25-50 µm, via intraarticular injection.

Another aspect provided herein is a method or formulation of any of thepreceding embodiments, wherein the formulation is delivered viainhalation as an aerosol.

Another aspect provided herein is a method or formulation of any of thepreceding embodiments, wherein the formulation is delivered viaintra-articular injection.

Another aspect provided herein is a method or formulation of any of thepreceding embodiments, wherein the formulation is delivered viaintramuscular injection.

In one embodiment, method or formulation of any one of the precedingembodiments wherein, the formulation is administered to the subject suchthat the antifibrotic agent (e.g., a relaxin) is administered to asubject at a dose between 1-2000 µg/kg body weight; or between 10-100µg/kg body weight; or between 100-200 µg/kg body weight; or between200-500 µg/kg body weight; or between 500-1000 µg/kg body weight; or25-75 µg/kg body weight; or 30-70 µg/kg body weight; or 40-60 µg/kg bodyweight; or between 1-10 µg/kg body weight; or between 1-5 µg/kg bodyweight; or between 4-8 µg/kg body weight; or about 2 µg/kg body weight;or about 5 µg/kg body weight; or about 10 µg/kg body weight; or about 20µg/kg body weight; or about 25 µg/kg body weight; or about 30 µg/kg bodyweight; or about 35 µg/kg body weight; or about 40 µg/kg body weight; orabout 45 µg/kg body weight; or about 50 µg/kg body weight; or about 55µg/kg body weight; or about 60 µg/kg body weight; or about 65 µg/kg bodyweight; or about 70 µg/kg body weight; or about 75 µg/kg body weight; orabout 100 µg/kg body weight; or about 200 µg/kg body weight; or about500 µg/kg body weight.

Administration

In some embodiments, methods of the invention comprise administering anagent e.g., relaxin or an analog, a fragment or a variant thereof to asubject using a depot. The terms “administer”, “administering” or“administration” include any method of delivery of agent into thesubject’s system or to a particular region in or on the subject. Forexample, relaxin or agent loaded depot may be administeredintravenously, intramuscularly, subcutaneously, intradermally,intranasally, orally, transcutaneously, mucosally, intraarticularly,periarticularly, intracapsularly, pericapsularly, intratendinously,peritendinously, intraligamentously, periligamentously, by pulmonaryinhalation or by ocular specific routes of administration. Administeringthe agent loaded depot can be performed by a number of people working inconcert and can include, for example, prescribing relaxin or an analog,a fragment or a variant thereof to be administered to a subject via adepot and/or providing instructions, directly or through another, totake the relaxin or an analog, a fragment or a variant thereof, eitherby self-delivery via a depot, e.g., as by oral delivery, subcutaneousdelivery, intravenous delivery through a central line, etc., or fordelivery by a trained professional, e.g., intra-articular delivery,intravenous delivery, intramuscular delivery, intratumoral delivery,etc.

In a preferred embodiment, the agent e.g., relaxin or an analog, afragment or a variant thereof is administered locally, e.g., directly toor into a joint of a subject using a depot. Local administration of theagent (e.g., relaxin) loaded depot by an intraarticular injection or bytopical application to the joint, or in the tissue surrounding the jointis advantageous because it allows delivery of a smaller dose of theagent to the subject and avoids the side-effects associated withsystemic delivery, such as back pain and joint pain.

For example, in a prior clinical investigation of relaxin for treatingheart failure, relaxin was dosed at 30 micrograms/kg/day for 2 dayssystemically (intravenous infusion), and did not meet the trial’sprimary endpoint of effectiveness. In another prior clinicalinvestigation of relaxin for treating scleroderma, relaxin was dosed at25 micrograms/kg/day for 24 weeks systemically (subcutaneous delivery),and did not meet the trial’s primary endpoint of effectiveness. Incontrast, formulations of the present disclosure may delivery relaxinlocally (opposed to systemically) at an effective dose of about 0.1microgram/kg/day for about 4 or for about 6 weeks, and may demonstrateclinical effectiveness.

In one embodiment, the agent e.g., relaxin loaded depot is administeredto the subject by an intraarticular injection. In one embodiment, theagent e.g., relaxin loaded depot is administered to the subject by anintraarticular, periarticular, intracapsular, pericapsular,intraligamentous, periligamentous, intratendinous, peritendinous,intraosteotendinous, or periosteotendinous injection (collectively“joint injections”), or combination thereof. In one embodiment, theagent e.g., relaxin loaded depot is administered to the subject via asingle joint injection. In one embodiment, the agent e.g., relaxinloaded depot is administered to the subject via multiple jointinjections. The multiple joint injections of the agent e.g., relaxinloaded depot may be administered to a subject at regularly spaced timeintervals, e.g., every day, every 2 days, every 3 days, every 4 days,every 5 days, every 6 days, every 7 days, every 8 days, every 9 days,every 10 days, every 11 days, every 12 days every 13 days or every 14days. A course of treatment consisting of multiple joint injections ofagent e.g., relaxin loaded depot may be repeated.

In one embodiment, the agent is administered to or near tendons,osteotendinous junctions, tendon-bone interfaces, entheses, ormuscle-tendon insertions. Such tissues may be selected from thefollowing tendinous tissues, among others:

-   Shoulder    -   Teres Minor Tendons (Rotator Cuff) Infraspinatus Tendons        Supraspinatus Tendons Subscapularis Tendons-   Elbow/Forearm    -   Deltoid Tendons Biceps Tendons Triceps Tendons Brachioradialis        Tendons Extensor Carpi Radialis Brevis Tendons Extensor Carpi        Radialis Longus Tendons Supinator Tendons-   Wrist    -   Flexor Carpi Radialis Tendons Flexor Carpi Ulnaris Tendons        Extensor Capri Radialis Tendons Extensor Carpi Radialis Brevis        Tendons-   Hip/Groin    -   Iliopsoas Tendons Obturator Internus Tendons Adductor Longus,        Brevis, and Magnus Tendons Gluteus Maximus and Gluteus Medius        Tendons Iliotibial Band-   Knee    -   Quadriceps Tendons Patellar Tendons Hamstring Tendons Sartorius        Tendons-   Ankle    -   Gastrocnemius Tendons Achilles Tendons Soleus Tendons Tibialis        Anterior Tendons Peroneus Longus Tendons-   Hand (Fingers)    -   Flexor Digitorum Longus Tendons Interosseus Tendons Flexor        Digitorum Profundus Tendons Abductor Digiti Minimi Tendons-   Hand (Thumb)    -   Opponens Pollicis Tendons Flexor Pollicis Tendons Extensor and        Abductor Pollicis Tendons-   Foot (Toes)    -   Flexor Hallucis Longus Tendons Flexor Digitorum Brevis Tendons        Lumbrical Tendons Abductor Hallucis Tendons Flexor Digitorum        Longus Tendons Abductor Digiti Minimi Tendons Plantar Fasciitis-   Back    -   Multifidus Tendons Quadratus Lumborum Tendons Longissmus        Thoracis Tendons Iliocostalis Tendons Spinalis Thoracis Tendons        Psoas Major Tendons

The joint injection of the agent e.g., relaxin loaded depot may beaccomplished by using a syringe with a needle suited for a jointinjection. A needle suitable for an joint injection may be selected fromthe group consisting of a 30G needle, a 29G needle, a 28G needle, a 27Gneedle, a 26sG needle, a 26G needle, a 25.5G needle, a 25sG needle, a25G needle, a 24.5G needle, a 24G needle, a 23.5G needle, a 23sG needle,a 23G needle, a 22.5G needle, a 22sG needle, a 22G needle, a 21.5Gneedle, a 21G needle, a 20.5G needle, a 20G needle, a 19.5G needle, a19G needle, a 18.5G needle and an 18G needle. In a specific embodiment,the agent e.g., relaxin loaded depot is administered via a 21G needle.

In another preferred embodiment, the agent e.g., relaxin loaded depotmay be administered to a subject topically, e.g., transcutaneously. Forexample, the agent e.g., relaxin loaded depot may be administered as agel, a cream, an ointment, a lotion, a drop, a suppository, a spray, aliquid or a powder composition that is applied topically to a joint,e.g., a finger joint.

In some embodiments, the agent e.g., relaxin loaded depot may beadministered to a subject during a medical procedure, e.g., a surgery,to treat or prevent a stiffened joint. Because stiffened joint mayresult from a surgery, administering relaxin during surgery may preventformation of a stiffened joint in a subject. In one embodiment, theagent e.g., relaxin loaded depot may be administered through a cannulaor an incision.

In another embodiment, the agent e.g., relaxin loaded depot may beadministered during an outpatient arthroscopic, fluoroscopic orultrasound guided procedure.

In a preferred embodiment, the agent e.g., relaxin loaded depot isadministered to the subject locally in as a sustained releaseformulation. Administering relaxin as a sustained release formulation isadvantageous because it avoids repeated injections and can deliver atherapeutic dose of the relaxin in a consistent and reliable manner, andover a desired period of time. Exemplary sustained release formulationsthat may be used to delivery polypeptides, are described in Vaishya etal., Expert. Opin. Drug Deliv. 2015, 12(3):415-40, the entire contentsof which are incorporated herein by reference.

Certain embodiments of the invention provide a solution to secondaryarthrofibrosis developed in neuromotor degenerative diseases by thelocal intra-articular delivery of relaxin-2, for example in a sustainedrelease formulation. In some embodiments, Relaxin-2 may reduce fibrosisin an in-vivo neuromotor degenerative arthrofibrosis large animal modelby inhibiting TGF-β1 signaling via the NO-sGC-cGMP pathway, therebydecreasing joint stiffness and increasing range of motion.

In some embodiments, the formulation is provided as a lyophilized powderfor resuspension or reconstitution with diluent at or near the point ofcare.

Clinically, the treating physician may inject a formulation as disclosedherein, such as relaxin-2 microparticle formulation, in the afflictedcontracted joints and periarticular tissues of patients with progressiveneuromotor degenerative conditions. These injections will take place inan office setting using anatomical landmarks or under ultrasoundguidance. The injections may be followed by a standard course ofphysical therapy. For more difficult joint injections (i.e., hip,spine), fluoroscopic guided injections can also be performed by anorthopedic surgeon or an interventional radiologist. In someembodiments, advantages may be: Elimination of surgery in a high-risk;Reduction of lifetime health care costs; Local injection of relaxin-2 inthe synovial joint space via standard office injection techniques;Minimization of dose as a result of local and not systemic delivery;reduction of off-target side effects and increased safety due to localdelivery.

In addition to the tissues around joint, the formulation can treatfibrosis in additional target organs that express the relaxin receptorthrough different routes of administration.

For lung fibrosis such as insterstitial lung disease, idiopathicpulmonary fibrosis, cystic fibrosis, hypertension, the formulation canbe administered by pulmonary inhalation or intranasally as a sustainedrelease formulation and may be provided a single injections or doses ora series of injections or doses.

For liver fibrosis such as hepatitis B or C, long-term alcohol abuse,non-alcoholic steatohepatitis, non-alcoholic fatty liver disease,Cholestasis, autoimmune hepatitis cirrhosis; kidney fibrosis such aschronic kidney disease, end-stage renal disease, renal interstitialfibrosis; and heart disease such as heart failure, myocardialinfarction, aortic stenosis, hypertrophic cardiomyopathy, theformulation can be administered intravenously, intramuscularly orintravenously (such as by catheter) as a sustained release formulationand may be provided a single injections or doses or a series ofinjections or doses.

For intestinal diseases such as Crohn’s disease, inflammatory boweldisease, enteropathies, and other intestinal fibrosis, the formulationscan be administered intranasally, orally, mucosally, as a sustainedrelease formulation and may be provided a single injections or doses ora series of injections or doses.

For skin conditions such as scleroderma, keloids, hypertrophic scars,cellulite, the formulations can be administered intramuscularly,subcutaneously, intradermally, or transcutaneously as a sustainedrelease formulation and may be provided a single injections or doses ora series of injections or doses.

For urogenital and gynecological conditions such as Peyronie’s diseaseand uterine fibroids, the formulations can be administeredtranscutaneously or transmucosally as a sustained release formulationand may be provided a single injections or doses or a series ofinjections or doses.

For ocular diseases such as Congenital Fibrosis of the ExtraocularMuscles, subretinal fibrosis, epiretinal fibrosis, corneal fibrosis, theformulation can be administered topically, by local ocularadministration (ie, subconjunctival, subretinal, intravitreal,retrobulbar, intracameral), or systemically (ie orally, intravenously,nasally) as a sustained release formulation and may be provided a singleinjections or doses or a series of injections or doses. Additionaldiseases and conditions suitable for treatment include Dupuytren’sDisease (the formation of a collagen cord in the palm that contracts andlimits range of motion of fingers) Peyronie’s Disease (excess ofinelastic collagen causes penis curvature; distorts erection), Canineand Human Lipomas encapsulated deposits of benign fatty tumors), UterineFibroids (benign tumors with significant co-morbidities), PlantarFibromatosis (pain and disability caused by the thickening of the feet’sdeep connective tissue), Capsular Contracture, Breast (post-surgicalcomplication that can deform the breast and cause pain), HypertrophicScars & Keloids (scars that form on the skin at site of injury),Dercum’s Disease (obesity and overly sensitive painful adipose tissue)Knee Arthrofibrosis (adhesions that form post-implant that may affectrange of motion), Urethral Strictures Narrowing (Narrowing of theurethra that affects urine flow).

The microparticles and agents described herein can be administered to asubject having or diagnosed as having a disease or disorder associatedwith fibrosis. In some embodiments, the methods described hereincomprise administering an effective amount of a microparticle or agentto a subject in order to alleviate at least one symptom of the diseaseor disorder. As used herein, “alleviating at least one symptom of thedisease or disorder associated with fibrosis” is ameliorating anycondition or symptom associated with the fibrotic disease or disorder.As compared with an equivalent untreated control, such reduction is byat least 5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95%, 99% or more asmeasured by any standard technique. A variety of means for administeringthe agents described herein to subjects are known to those of skill inthe art. In one embodiment of any of the aspects, the agent isadministered systemically or locally (e.g., to the brain, or otheraffected organ, e.g., the colon).

In one embodiment of any of the aspects, the agent is administeredintravenously. In one embodiment of any of the aspects, the agent isadministered continuously, in intervals, or sporadically. The route ofadministration of the agent will be optimized for the type of agentbeing delivered (e.g., an antibody, a small molecule, an RNAi), and canbe determined by a skilled practitioner.

The term “effective amount” as used herein refers to the amount of amicroparticle or anti-fibrotic agent as described herein can beadministered to a subject having or diagnosed as having a disease ordisorder associated with fibrosis needed to alleviate at least one ormore symptom of the disease or disorder. The term “therapeuticallyeffective amount” therefore refers to an amount of an agent that issufficient to provide a particular anti-disease or disorder effect whenadministered to a typical subject. An effective amount as used herein,in various contexts, would also include an amount of a microparticle oragent sufficient to delay the development of a symptom of the disease ordisorder, alter the course of a symptom of the disease or disorder(e.g., inflammation, stiffening of a joint, pain, loss of mobility,difficulty breathing), or reverse a symptom of the disease or disorder(e.g., inflammation, stiffening of a joint, pain, loss of mobility,difficulty breathing). Thus, it is not generally practicable to specifyan exact “effective amount”. However, for any given case, an appropriate“effective amount” can be determined by one of ordinary skill in the artusing only routine experimentation.

In one embodiment of any of the aspects, the agent is administeredcontinuously (e.g., at constant levels over a period of time).Continuous administration of an agent can be achieved, e.g., byepidermal patches, continuous release formulations, or on-bodyinjectors.

Effective amounts, toxicity, and therapeutic efficacy can be evaluatedby standard pharmaceutical procedures in cell cultures or experimentalanimals. The dosage can vary depending upon the dosage form employed andthe route of administration utilized. The dose ratio between toxic andtherapeutic effects is the therapeutic index and can be expressed as theratio LD50/ED50. Compositions and methods that exhibit large therapeuticindices are preferred. A therapeutically effective dose can be estimatedinitially from cell culture assays. Also, a dose can be formulated inanimal models to achieve a circulating plasma concentration range thatincludes the IC50 (i.e., the concentration of the agent, which achievesa half-maximal inhibition of symptoms) as determined in cell culture, orin an appropriate animal model. Levels in plasma can be measured, forexample, by high performance liquid chromatography. The effects of anyparticular dosage can be monitored by a suitable bioassay, e.g.,measuring neurological function, or blood work, among others. The dosagecan be determined by a physician and adjusted, as necessary, to suitobserved effects of the treatment. Dosage

“Unit dosage form” as the term is used herein refers to a dosage forsuitable one administration. By way of example a unit dosage form can bean amount of therapeutic disposed in a delivery device, e.g., a syringeor intravenous drip bag. In one embodiment of any of the aspects, a unitdosage form is administered in a single administration. In another,embodiment more than one unit dosage form can be administeredsimultaneously.

The dosage of the agent as described herein can be determined by aphysician and adjusted, as necessary, to suit observed effects of thetreatment. With respect to duration and frequency of treatment, it istypical for skilled clinicians to monitor subjects in order to determinewhen the treatment is providing therapeutic benefit, and to determinewhether to administer further cells, discontinue treatment, resumetreatment, or make other alterations to the treatment regimen. Thedosage should not be so large as to cause adverse side effects, such ascytokine release syndrome. Generally, the dosage will vary with the age,condition, and sex of the patient and can be determined by one of skillin the art. The dosage can also be adjusted by the individual physicianin the event of any complication.

The effective dose can be estimated initially from cell culture assays.A dose can be formulated in animals. Generally, the compositions areadministered so that a compound of the invention herein is used or givenat a dose from 1 µg/kg to 1000 mg/kg; 1 µg/kg to 500 mg/kg; 1 µg/kg to150 mg/kg, 1 µg/kg to 100 mg/kg, 1 µg/kg to 50 mg/kg, 1 µg/kg to 20mg/kg, 1 µg/kg to 10 mg/kg, 1 µg/kg to 1 mg/kg, 100 µg/kg to 100 mg/kg,100 µg/kg to 50 mg/kg, 100 µg/kg to 20 mg/kg, 100 µg/kg to 10 mg/kg, 100µg/kg to 1 mg/kg, 1 mg/kg to 100 mg/kg, 1 mg/kg to 50 mg/kg, 1 mg/kg to20 mg/kg, 1 mg/kg to 10 mg/kg, 10 mg/kg to 100 mg/kg, 10 mg/kg to 50mg/kg, or 10 mg/kg to 20 mg/kg. It is to be understood that ranges givenhere include all intermediate ranges, for example, the range 1 mg/kg to10 mg/kg includes 1 mg/kg to 2 mg/kg, 1 mg/kg to 3 mg/kg, 1 mg/kg to 4mg/kg, 1 mg/kg to 5 mg/kg, 1 mg/kg to 6 mg/kg, 1 mg/kg to 7 mg/kg, 1mg/kg to 8 mg/kg, 1 mg/kg to 9 mg/kg, 2 mg/kg to 10 mg/kg, 3 mg/kg to 10mg/kg, 4 mg/kg to 10 mg/kg, 5 mg/kg to 10 mg/kg, 6 mg/kg to 10 mg/kg, 7mg/kg to 10 mg/kg, 8 mg/kg to 10 mg/kg, 9 mg/kg to 10 mg/kg, and thelike. Further contemplated is a dose (either as a bolus or continuousinfusion) of about 0.1 mg/kg to about 10 mg/kg, about 0.3 mg/kg to about5 mg/kg, or 0.5 mg/kg to about 3 mg/kg. It is to be further understoodthat the ranges intermediate to those given above are also within thescope of this invention, for example, in the range 1 mg/kg to 10 mg/kg,for example use or dose ranges such as 2 mg/kg to 8 mg/kg, 3 mg/kg to 7mg/kg, 4 mg/kg to 6 mg/kg, and the like.

Combinational Therapy

In one embodiment of any of the aspects, the microparticle or agentdescribed herein is used as a monotherapy. In another embodiment of anyof the aspects, the microparticle or agent described herein can be usedin combination with other known agents and therapies (i.e. cotherapies)for a disease, condition, or disorder, such as a disease, condition, ordisorder associated with fibrosis. Administered “in combination,” asused herein, means that two (or more) different treatments are deliveredto the subject during the course of the subject’s affliction with thedisorder, e.g., the two or more treatments are delivered after thesubject has been diagnosed with the disorder (a fibrotic disease ordisorder) and before the disorder has been cured or eliminated ortreatment has ceased for other reasons. In some embodiments, thedelivery of one treatment is still occurring when the delivery of thesecond begins, so that there is overlap in terms of administration. Thisis sometimes referred to herein as “simultaneous” or “concurrentdelivery.” In other embodiments, the delivery of one treatment endsbefore the delivery of the other treatment begins. In some embodimentsof either case, the treatment is more effective because of combinedadministration. For example, the second treatment is more effective,e.g., an equivalent effect is seen with less of the second treatment, orthe second treatment reduces symptoms to a greater extent, than would beseen if the second treatment were administered in the absence of thefirst treatment, or the analogous situation is seen with the firsttreatment. In some embodiments, delivery is such that the reduction in asymptom, or other parameter related to the disorder is greater than whatwould be observed with one treatment delivered in the absence of theother. The effect of the two treatments can be partially additive,wholly additive, or greater than additive. The delivery can be such thatan effect of the first treatment delivered is still detectable when thesecond is delivered. The agents described herein and the at least oneadditional therapy can be administered simultaneously, in the same or inseparate compositions, or sequentially. For sequential administration,the agent described herein can be administered first, and the additionalagent can be administered second, or the order of administration can bereversed. The agent and/or other therapeutic agents, procedures ormodalities can be administered during periods of active disorder, orduring a period of remission or less active disease. The microparticleor agent of this disclosure can be administered before anothertreatment, concurrently with the treatment, posttreatment, or duringremission of the disorder.

When administered in combination, the agent and the additional agent(e.g., second or third agent), or all, can be administered in an amountor dose that is higher, lower or the same as the amount or dosage ofeach agent used individually, e.g., as a monotherapy. In certainembodiments, the administered amount or dosage of the agent, theadditional agent (e.g., second or third agent), or all, is lower (e.g.,at least 20%, at least 30%, at least 40%, or at least 50%) than theamount or dosage of each agent used individually. In other embodiments,the amount or dosage of agent, the additional agent (e.g., second orthird agent), or all, that results in a desired effect (e.g., treatmentof a fibrotic disease or disorder) is lower (e.g., at least 20%, atleast 30%, at least 40%, or at least 50% lower) than the amount ordosage of each agent individually required to achieve the sametherapeutic effect.

In some embodiments, the cotherapy is a drug, such as aspirin,acetaminophen, non-steroidal anti-inflammatory drugs, steroids, nerveblockers, and analgesic drugs common in the art.

In some embodiments, the cotherapy is a drug for muscular dystrophies,including but not limited to deflazacourt, eteplirsen, casimersen,golodirsen, ataluren, givinostat, viltolarsen, pamrevlumab, SRP-9001,SRP-5051, DS-5141B, SCAAV9.U7.ACCA, PF-06939926, SGT-001, or AT702.

In some embodiments, the cotherapy is a drug for spinal muscularatrophy, including but not limited to Spinraza, Zolgensma, Evrysdi,SRK-015, CK-2127107, LMI070, AVXS-101, BIIB110, or p38aMAPK inhibitors.

In some embodiments, the cotherapy is a drug for cerebral palsy, stroke,traumatic brain injury, or peripheral nerve injury, including but notlimited to anticholinergics such as Benztropine mesylate,Carbidopa-levodopa (Sinemet), Glycopyrrolate (Robinul), Procyclidinehydrochloride (Kemadrin), and Trihexyphenidyl hydrochloride;anticonvulsants such as Gabapentin (Neurontin), Lamotrigine (Lamictal),Oxcarbazepine (Trileptal), Topiramate (Topamax), and Zonisamide(Zonegran); or antispastics i.e. muscle relaxants such as Baclofen,Botulinum toxin, Diazepam (Valium(R)), Dantrolene, Flexeril(Cyclobenzadrine), Dantrium (Dantrolene), or Tizanidine.

In some embodiments, the cotherapy is physical therapy.

In some embodiments, the cotherapy is a surgical intervention, includingbut not limited to surgical release, capsular release, or surgicalrepair.

In some embodiments, the cotherapy is an energy-based technique,including but not limited to radiofrequency energy application e.g.radiofrequency ablation, thermal energy application or removal e.g.cryoablation, sonic energy application e.g. ultrasound-based therapeutictechniques, electrical energy application e.g. transcutaneous electricalnerve stimulation (TENs), or other electromagnetic energy application orremoval methods such as light exposure.

In some embodiments, the cotherapy is an exoskeleton designed to assistambulation or other motion in patients with ambulatory or othermotion-based dysfunction.

Parenteral Dosage Forms

Parenteral dosage forms of an agents described herein can beadministered to a subject by various routes, including, but not limitedto, subcutaneous, intravenous (including bolus injection),intramuscular, and intraarterial. Since administration of parenteraldosage forms typically bypasses the patient’s natural defenses againstcontaminants, parenteral dosage forms are preferably sterile or capableof being sterilized prior to administration to a patient. Examples ofparenteral dosage forms include, but are not limited to, solutions readyfor injection, dry products ready to be dissolved or suspended in apharmaceutically acceptable vehicle for injection, suspensions ready forinjection, controlled-release parenteral dosage forms, and emulsions.

The phrases “parenteral administration” and “administered parenterally”as used herein means modes of administration other than enteral andtopical administration, usually by injection, and includes, withoutlimitation, intravenous, intramuscular, perimuscular, intraarterial,intrathecal, intraventricular, intracapsular, pericapsular,intraorbital, intracardiac, intradermal, peridermal, intraperitoneal,transtracheal, subcutaneous, subcuticular, intraarticular,periarticular, sub capsular, subarachnoid, intraspinal, intracerebrospinal, and intrasternal injection, infusion and other injection orinfusion techniques, without limitation. Without limitations, oraladministration can be in the form of solutions, suspensions, tablets,pills, capsules, sustained-release formulations, oral rinses, powdersand the like. Suitable vehicle solutions that can be used to provideparenteral dosage forms of the invention are well known to those skilledin the art. As used herein, the phrase “vehicle solutions” include,without limitation: sterile water; water for injection USP; salinesolution; sodium carboxymethylcellulose; glucose solution; aqueousvehicles such as but not limited to, sodium chloride injection, Ringer’sinjection, dextrose injection, dextrose and sodium chloride injection,and lactated Ringer’s injection; water-miscible vehicles such as, butnot limited to, ethyl alcohol, polyethylene glycol, and propyleneglycol; and non-aqueous vehicles such as, but not limited to, corn oil,cottonseed oil, peanut oil, sesame oil, ethyl oleate, isopropylmyristate, and benzyl benzoate.

Efficacy

The efficacy of an agents described herein, e.g., for the treatment of adisease or disorder associated with fibrosis, can be determined by theskilled practitioner. However, a treatment is considered “effectivetreatment,” as the term is used herein, if one or more of the signs orsymptoms of the fibrotic disease are altered in a beneficial manner,other clinically accepted symptoms are improved, or even ameliorated, ora desired response is induced e.g., by at least 10% following treatmentaccording to the methods described herein. Efficacy can be assessed, forexample, by measuring a marker, indicator, symptom, and/or the incidenceof a condition treated according to the methods described herein or anyother measurable parameter appropriate. Efficacy can also be measured bya failure of an individual to worsen as assessed by hospitalization, orneed for medical interventions (i.e., progression of the disease ordisorder, as measured by symptoms of the disease or disorder). Methodsof measuring these indicators are known to those of skill in the artand/or are described herein.

Efficacy can be assessed in animal models of a condition describedherein, for example, a mouse model or an appropriate animal model of afibrotic disease or disorder, as the case may be. When using anexperimental animal model, efficacy of treatment is evidenced when astatistically significant change in a marker is observed.

In some embodiments, efficacy of treatment includes the minimization offoreign-body-response or immune reaction after administration. Forexample, the administration of a vehicle control formulation (e.g. aPLGA microparticle containing no therapeutic agent) may elicitmacrophage and immune activation as well as inflammation, whereas theadministration of a formulation described by the present disclosure mayellict a lower immune response or entirely abrogate the elicited immuneresponse at any point throughout the treatment and assessment afteradministration.

In some embodiments, foreign body response resulting from administrationof a formulation described by the present disclosure may be reduced orabrogated compared to foreign body response resulting fromadministration of a PLGA microparticle containing a steroid as thetherapeutic agent.

The inventions illustratively described herein may be practiced in theabsence of any element or elements, limitation or limitations which isnot specifically disclosed herein. The terms and expressions which havebeen employed are used as terms of description and not of limitation,and there is no intention that in the use of such terms and expressionsof excluding any equivalents of the features shown and described orportions thereof, but it is recognized that various modifications arepossible within the scope of the invention claimed. Thus, it should beunderstood that although the present inventions have been specificallydisclosed by preferred embodiments and optional features, modificationand variation of the concepts herein disclosed may be resorted to bythose skilled in the art, and that such modifications and variations areconsidered to be within the scope of the inventions as defined by theappended embodiments and elsewhere in the invention.

The contents of the articles, patents, and patent applications, and allother documents and electronically available information mentioned orcited herein, are hereby incorporated by reference in their entirety tothe same extent as if each individual publication was specifically andindividually indicated to be incorporated by reference. Applicantsreserve the right to physically incorporate into this application anyand all materials and information from any such articles, patents,patent applications, or other documents.

The term “about” as used herein means in quantitative terms plus orminus 10%. For example, “About 3%” would encompass 2.7-3.3% and “About10%” would encompass 9-11%”. Moreover, where “about” is used herein inconjunction with a quantitative term it is understood that in additionto the value plus or minus 10%, the exact value of the quantitative termis also contemplated and described-for example, the term “about 3%”expressly contemplates, describes and includes exactly 3%.

The inventions illustratively described herein may suitably be practicedin the absence of any element or elements, limitation or limitations,not specifically disclosed herein. Thus, for example, the terms“comprising”, “including,” containing”, etc. shall be read expansivelyand without limitation. Additionally, the terms and expressions employedherein have been used as terms of description and not of limitation, andthere is no intention in the use of such terms and expressions ofexcluding any equivalents of the features shown and described orportions thereof, but it is recognized that various modifications arepossible within the scope of the inventions claimed. Thus, it should beunderstood that although the present inventions have been specificallydisclosed by preferred embodiments and optional features, modificationand variation of the inventions embodied therein herein disclosed may beresorted to by those skilled in the art, and that such modifications andvariations are considered to be within the scope of various aspects andembodiments of inventions contemplated herein.

Certain aspects and embodiments of the invention and inventions havebeen described broadly and generically herein. Each of the narrowerspecies and subgeneric groupings falling within the generic inventionalso form part of some aspects and embodiments of inventionscontemplated herein. This includes the generic description of inventionswith a proviso or negative limitation removing any subject matter fromthe genus, regardless of whether or not the excised material isspecifically recited herein.

In addition, where features or aspects of the invention are described interms of Markush groups, those skilled in the art will recognize thatsome aspects and embodiments of inventions contemplated herein are alsothereby described in terms of any individual member or subgroup ofmembers of the Markush group.

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the inventions. It should beunderstood that various alternatives to the embodiments of theinventions described herein may be employed in practicing theinventions. It is intended that the following embodiments define thescope of the inventions and that methods and structures within the scopeof these embodiments and their equivalents be covered thereby.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which these inventions belong. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the inventions, it will be understood thatmodifications and variations are encompassed within the spirit and scopeof the instant invention. The preferred methods and materials are nowdescribed.

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.In the case of conflict, the specification, including definitions, willcontrol.

Some embodiments of the technology described herein can be definedaccording to any of the following numbered paragraphs:

-   1. A formulation comprising microparticles comprising an aliphatic    polyester and an antifibrotic agent, wherein said microparticles    have a diameter of 1-100 µm.-   2. A formulation comprising microparticles comprising an aliphatic    polyester and an antifibrotic agent, wherein the antifibrotic agent    is relaxin and is present in an amount that is 0.01-33% of total    mass.-   3. A formulation comprising microparticles comprising an aliphatic    polyester and an antifibrotic agent, wherein said aliphatic    polyester is of molecular weight 10,000-200,000 daltons.-   4. A formulation comprising microparticles comprising an aliphatic    polyester, a vinyl polymer and an antifibrotic agent.-   5. A formulation comprising microparticles comprising an aliphatic    polyester, a vinyl polymer and an antifibrotic agent, wherein said    microparticles have a diameter of 1-100 µm.-   6. A formulation comprising microparticles comprising an aliphatic    polyester, a vinyl polymer and an antifibrotic agent, wherein the    antifibrotic agent is relaxin and is present in an amount that is    0.01-33% of total mass.-   7. A formulation comprising microparticles comprising an aliphatic    polyester, a vinyl polymer and an antifibrotic agent, wherein said    aliphatic polyester is of molecular weight 10,000-200,000 daltons.-   8. A formulation comprising PLGA microparticles comprising an    aliphatic polyester and an antifibrotic agent, wherein said    microparticles have a diameter of 1-100µm.-   9. A formulation comprising PLGA microparticles comprising an    aliphatic polyester and an antifibrotic agent, wherein the    antifibrotic agent is relaxin and is present in an amount that is    0.01-33% of total mass.-   10. A formulation comprising PLGA microparticles comprising an    aliphatic polyester and an antifibrotic agent, wherein said    aliphatic polyester is of molecular weight 10,000-200,000 daltons.-   11. A formulation comprising PLGA microparticles comprising an    aliphatic polyester, a vinyl polymer and an antifibrotic agent,    wherein said microparticles have a diameter of 1-50 µm.-   12. A formulation comprising PLGA microparticles comprising an    aliphatic polyester, a vinyl polymer and an antifibrotic agent,    wherein the antifibrotic agent is relaxin and is present in an    amount that is 0.1-10% of total mass.-   13. A formulation comprising PLGA microparticles comprising an    aliphatic polyester, a vinyl polymer and an antifibrotic agent,    wherein said aliphatic polyester is of molecular weight    10,000-200,000 daltons.-   14. The formulation of any one of the preceding paragraphs, wherein    said antifibrotic agent is an agonist of the receptor RXFP1.-   15. The formulation of any one of the preceding paragraphs, wherein    said antifibrotic agent is human relaxin-2 or an analog or variant.-   16. The formulation of any one of the preceding paragraphs, wherein    the aliphatic polyester is poly-lactide-co-glycolide.-   17. The formulation of any one of the preceding paragraphs, wherein    the aliphatic polyester is polycaprolactone.-   18. The formulation of any of the preceding paragraphs, wherein said    aliphatic polyester is of molecular weight 10,000-200,000 daltons.-   19. The formulation of any of the preceding paragraphs, wherein said    aliphatic polyester is of molecular weight 10,000-150,000 daltons;    or 25,000-125,000 daltons; or 40,00-100,000 daltons.-   20. The formulation of any of the preceding paragraphs, wherein said    aliphatic polyester is of molecular weight 10,000-30,000 daltons.-   21. The formulation of any of the preceding paragraphs, wherein said    aliphatic polyester is of molecular weight 30,000-50,000 daltons.-   22. The formulation of any of the preceding paragraphs, wherein said    aliphatic polyester is of molecular weight 50,000-70,000 daltons.-   23. The formulation of any of the preceding paragraphs, wherein said    aliphatic polyester is of molecular weight 70,000-90,000 daltons.-   24. The formulation of any of the preceding paragraphs, wherein said    aliphatic polyester is of molecular weight 90,000-120,000 daltons.-   25. The formulation of any of the preceding paragraphs, wherein said    aliphatic polyester is of molecular weight 120,000-150,000 daltons.-   26. The formulation of any of the preceding paragraphs, wherein said    aliphatic polyester is terminated by an ester functional group.-   27. The formulation of any of the preceding paragraphs, wherein said    aliphatic polyester is terminated by an alkyl-ester functional    group.-   28. The formulation of any of the preceding paragraphs, wherein said    aliphatic polyester is terminated by a carboxylic acid functional    group.-   29. The formulation of any of the preceding paragraphs, wherein said    formulation comprises a vinyl polymer that is poly(vinyl alcohol).-   30. The formulation of any of the preceding paragraphs, wherein said    formulation comprises a vinyl polymer that is poly(pyrrolidone).-   31. The formulation of any of the preceding paragraphs, wherein said    formulation comprises a vinyl polymer that is of molecular weight    10,000-200,000 daltons.-   32. The formulation of any of the preceding paragraphs, wherein said    formulation comprises a vinyl polymer that is of molecular weight    molecular weight 10,000-150,000 daltons; or 25,000-125,000 daltons;    or 40,00-100,000 daltons.-   33. The formulation of any of the preceding paragraphs, wherein said    formulation comprises a vinyl polymer that is of molecular weight    molecular weight 30,000-50,000 daltons.-   34. The formulation of any of the preceding paragraphs, wherein said    formulation comprises a vinyl polymer that is of molecular weight    molecular weight 50,000-70,000 daltons.-   35. The formulation of any of the preceding paragraphs, wherein said    formulation comprises a vinyl polymer that is of molecular weight    molecular weight 70,000-90,000 daltons.-   36. The formulation of any of the preceding paragraphs, wherein said    formulation comprises a vinyl polymer that is of molecular weight    molecular weight 90,000-120,000 daltons.-   37. The formulation of any of the preceding paragraphs, wherein the    diameter of said microparticles is 1-100 µm.-   38. The formulation of any of the preceding paragraphs, wherein the    diameter of said microparticles is 1-75 µm; or 1-50 µm; or 5-50 µm;    or 25-50 µm; or 30-50 µm; or 40-50 µm.-   39. The formulation of any of the preceding paragraphs, wherein the    diameter of said microparticles is 5-10 µm.-   40. The formulation of any of the preceding paragraphs, wherein the    diameter of said microparticles is 5-8 µm.-   41. The formulation of any of the preceding paragraphs, wherein the    diameter of said microparticles is 8-12 µm.-   42. The formulation of any of the preceding paragraphs, wherein the    diameter of said microparticles is 12-18 µm.-   43. The formulation of any of the preceding paragraphs, wherein the    diameter of said microparticles is 18-25 µm.-   44. The formulation of any of the preceding paragraphs, wherein the    diameter of said microparticles is 25-35 µm.-   45. The formulation of any of the preceding paragraphs, wherein the    diameter of said microparticles is 35-45 µm.-   46. The formulation of any of the preceding paragraphs, wherein the    diameter of said microparticles is 45-50 µm.-   47. The formulation of any of the preceding paragraphs, wherein the    diameter of said microparticles is about 1 µm.-   48. The formulation of any of the preceding paragraphs, wherein the    diameter of said microparticles is about 2 µm.-   49. The formulation of any of the preceding paragraphs, wherein the    diameter of said microparticles is about 3 µm.-   50. The formulation of any of the preceding paragraphs, wherein the    diameter of said microparticles is about 4 µm.-   51. The formulation of any of the preceding paragraphs, wherein the    diameter of said microparticles is about 5 µm.-   52. The formulation of any of the preceding paragraphs, wherein the    diameter of said microparticles is about 6 µm.-   53. The formulation of any of the preceding paragraphs, wherein the    diameter of said microparticles is about 7 µm.-   54. The formulation of any of the preceding paragraphs, wherein the    diameter of said microparticles is about 8 µm.-   55. The formulation of any of the preceding paragraphs, wherein the    diameter of said microparticles is about 9 µm.-   56. The formulation of any of the preceding paragraphs, wherein the    diameter of said microparticles is about 10 µm.-   57. The formulation of any of the preceding paragraphs, wherein the    diameter of said microparticles is about 15 µm.-   58. The formulation of any of the preceding paragraphs, wherein the    diameter of said microparticles is about 20 µm.-   59. The formulation of any of the preceding paragraphs, wherein the    diameter of said microparticles is about 25 µm.-   60. The formulation of any of the preceding paragraphs, wherein the    diameter of said microparticles is about 30 µm.-   61. The formulation of any of the preceding paragraphs, wherein the    diameter of said microparticles is about 35 µm.-   62. The formulation of any of the preceding paragraphs, wherein the    diameter of said microparticles is about 40 µm.-   63. The formulation of any of the preceding paragraphs, wherein the    diameter of said microparticles is about 45 µm.-   64. The formulation of any of the preceding paragraphs, wherein the    diameter of said microparticles is about 50 µm.-   65. The formulation of any of the preceding paragraphs, wherein the    diameter of said microparticles is about 75 µm.-   66. The formulation of any of the preceding paragraphs, wherein the    diameter of said microparticles is about 100 µm.-   67. The formulation of any of the preceding paragraphs, wherein the    diameter of said microparticles is about 150 µm.-   68. The formulation of any of the preceding paragraphs, wherein the    diameter of said microparticles is about 200 µm.-   69. The formulation of any of the preceding paragraphs, wherein said    aliphatic polyester is poly-lactide-co-glycolide with a molar ratio    of 15:85 - 25:75, lactide:glycolide.-   70. The formulation of any of the preceding paragraphs, wherein said    aliphatic polyester is poly-lactide-co-glycolide with a molar ratio    of 25:75 - 35:65, lactide:glycolide.-   71. The formulation of any of the preceding paragraphs, wherein said    aliphatic polyester is poly-lactide-co-glycolide with a molar ratio    of 35:65 - 45:55, lactide:glycolide.-   72. The formulation of any of the preceding paragraphs, wherein said    aliphatic polyester is poly-lactide-co-glycolide with a molar ratio    of 45:55 - 55:45, lactide:glycolide.-   73. The formulation of any of the preceding paragraphs, wherein said    aliphatic polyester is poly-lactide-co-glycolide with a molar ratio    of 55:45 - 65:35, lactide:glycolide.-   74. The formulation of any of the preceding paragraphs, wherein said    aliphatic polyester is poly-lactide-co-glycolide with a molar ratio    of 65:35 - 75:25, lactide:glycolide.-   75. The formulation of any of the preceding paragraphs, wherein said    aliphatic polyester is poly-lactide-co-glycolide with a molar ratio    of 75:25 - 85:15, lactide:glycolide.-   76. The formulation of any of the preceding paragraphs, wherein said    aliphatic polyester is poly-lactide-co-glycolide with a molar ratio    of about 50:50, lactide:glycolide.-   77. The formulation of any of the preceding paragraphs, wherein said    aliphatic polyester is poly-lactide-co-glycolide with a molar ratio    of about 45:55, lactide:glycolide.-   78. The formulation of any of the preceding paragraphs, wherein said    aliphatic polyester is poly-lactide-co-glycolide with a molar ratio    of about 55:45, lactide:glycolide.-   79. The formulation of any of the preceding paragraphs, wherein said    aliphatic polyester is poly-lactide-co-glycolide with a molar ratio    of about 40:60, lactide:glycolide.-   80. The formulation of any of the preceding paragraphs, wherein said    aliphatic polyester is poly-lactide-co-glycolide with a molar ratio    of about 60:40, lactide:glycolide.The formulation of any of the    preceding paragraphs, wherein the formulation comprises a vinyl    polymer that is 0.01-0.1% of total mass.-   81. The formulation of any of the preceding paragraphs, wherein the    formulation comprises a vinyl polymer that is 0.1-0.3% of total    mass.-   82. The formulation of any of the preceding paragraphs, wherein the    formulation comprises a vinyl polymer that is 0.2-0.9% of total    mass.-   83. The formulation of any of the preceding paragraphs, wherein the    formulation comprises a vinyl polymer that is 0.3-0.7% of total    mass.-   84. The formulation of any of the preceding paragraphs, wherein the    formulation comprises a vinyl polymer that is 0.4-0.6% of total    mass.-   85. The formulation of any of the preceding paragraphs, wherein the    formulation comprises a vinyl polymer that is 0.3-0.6% of total    mass.-   86. The formulation of any of the preceding paragraphs, wherein the    formulation comprises a vinyl polymer that is 0.6-1.0% of total    mass.-   87. The formulation of any of the preceding paragraphs, wherein the    formulation comprises a vinyl polymer that is 1.0-5.0% of total    mass.-   88. The formulation of any of the preceding paragraphs, wherein the    formulation comprises a vinyl polymer that is 5.0-10.0% of total    mass.-   89. The formulation of any of the preceding paragraphs, wherein the    formulation comprises a vinyl polymer that is 10.0-30.0% of total    mass.-   90. The formulation of any of the preceding paragraphs, wherein the    formulation comprises a vinyl polymer that is about 0.1% of total    mass.-   91. The formulation of any of the preceding paragraphs, wherein the    formulation comprises a vinyl polymer that is about 0.2% of total    mass.-   92. The formulation of any of the preceding paragraphs, wherein the    formulation comprises a vinyl polymer that is about 0.3% of total    mass.-   93. The formulation of any of the preceding paragraphs, wherein the    formulation comprises a vinyl polymer that is about 0.4% of total    mass.-   94. The formulation of any of the preceding paragraphs, wherein the    formulation comprises a vinyl polymer that is about 0.5% of total    mass.-   95. The formulation of any of the preceding paragraphs, wherein the    formulation comprises a vinyl polymer that is about 0.6% of total    mass.-   96. The formulation of any of the preceding paragraphs, wherein the    formulation comprises a vinyl polymer that is about 0.7% of total    mass.-   97. The formulation of any of the preceding paragraphs, wherein the    formulation comprises a vinyl polymer that is about 0.8% of total    mass.-   98. The formulation of any of the preceding paragraphs, wherein the    formulation comprises a vinyl polymer that is about 0.9% of total    mass.-   99. The formulation of any of the preceding paragraphs, wherein the    formulation comprises a vinyl polymer that is about 10% of total    mass.-   100. The formulation of any of the preceding paragraphs, wherein the    formulation comprises a vinyl polymer that is about 15% of total    mass.-   101. The formulation of any of the preceding paragraphs, wherein the    formulation comprises a vinyl polymer that is about 20% of total    mass.-   102. The formulation of any of the preceding paragraphs, wherein the    formulation comprises a vinyl polymer that is about 25% of total    mass.-   103. The formulation of any of the preceding paragraphs, wherein the    formulation comprises a vinyl polymer that is about 30% of total    mass.-   104. The formulation of any of the preceding paragraphs, wherein    said antifibrotic agent is 0.005-5% of the total formulation mass.-   105. The formulation of any of the preceding paragraphs, wherein    said antifibrotic agent is 0.01-10%; 0.01-33%; or 0.1-5% of the    total formulation mass; or 0.2-4% of the total formulation mass; or    0.3-3% of the total formulation mass; or 0.5-2% of the total    formulation mass; or 0.5-1.5% of the total formulation mass; or    0.5-3% of the total formulation mass; or 1-2% of the total    formulation mass; or 1-5% of the total formulation mass; or 3-7% of    the total formulation mass; or 5-10% of the total formulation mass.-   106. The formulation of any of the preceding paragraphs, wherein    said antifibrotic agent is 0.005-0.01% of the total formulation    mass.-   107. The formulation of any of the preceding paragraphs, wherein    said antifibrotic agent is 0.01-0.05% of the total formulation mass.-   108. The formulation of any of the preceding paragraphs, wherein    said antifibrotic agent is 0.05-0.1% of the total formulation mass.-   109. The formulation of any of the preceding paragraphs, wherein    said antifibrotic agent is 0.1-0.5% of the total formulation mass.-   110. The formulation of any of the preceding paragraphs, wherein    said antifibrotic agent is 0.5-1.0% of the total formulation mass.-   111. The formulation of any of the preceding paragraphs, wherein    said antifibrotic agent is 1.0-2.5% of the total formulation mass.-   112. The formulation of any of the preceding paragraphs, wherein    said antifibrotic agent is 2.5-5.0% of the total formulation mass.-   113. The formulation of any of the preceding paragraphs, wherein    said antifibrotic agent is about 0.25% of the total formulation    mass.-   114. The formulation of any of the preceding paragraphs, wherein    said antifibrotic agent is about 0.5% of the total formulation mass.-   115. The formulation of any of the preceding paragraphs, wherein    said antifibrotic agent is about 0.75% of the total formulation    mass.-   116. The formulation of any of the preceding paragraphs, wherein    said antifibrotic agent is about 1% of the total formulation mass.-   117. The formulation of any of the preceding paragraphs, wherein    said antifibrotic agent is about 1.25% of the total formulation    mass.-   118. The formulation of any of the preceding paragraphs, wherein    said antifibrotic agent is about 1.5% of the total formulation mass.-   119. The formulation of any of the preceding paragraphs, wherein    said antifibrotic agent is about 1.75% of the total formulation    mass.-   120. The formulation of any of the preceding paragraphs, wherein    said antifibrotic agent is about 2% of the total formulation mass.-   121. The formulation of any of the preceding paragraphs, wherein    said antifibrotic agent is about 2.5% of the total formulation mass.-   122. The formulation of any of the preceding paragraphs, wherein    said antifibrotic agent is about 3% of the total formulation mass.-   123. The formulation of any of the preceding paragraphs, wherein    said antifibrotic agent is about 5% of the total formulation mass.-   124. The formulation of any of the preceding paragraphs, wherein    said antifibrotic agent is a relaxin.-   125. The formulation of any of the preceding paragraphs, wherein    said formulation comprises PLGA microparticles with a PLGA molar    ratio that is about 50:50 lactide:glycolide, a relaxin loaded at    about 1% by weight of the microparticles, and PVA in a concentration    of about 0.5% by weight.-   126. The formulation of any of the preceding paragraphs, wherein    said formulation comprises PLGA microparticles with a PLGA molar    ratio that is about 50:50 lactide:glycolide, a relaxin loaded at    about 1% by weight of the microparticles and PVA in a concentration    of about 0.0% by weight-   127. The formulation of any of the preceding paragraphs, wherein    said formulation comprises PLGA microparticles with a PLGA molar    ratio that is about 60:40 lactide:glycolide, a relaxin loaded at    about 1% by weight of the microparticles, and PVA in a concentration    of about 0.5% by weight.-   128. The formulation of any of the preceding paragraphs, wherein    said formulation comprises PLGA microparticles with a PLGA molar    ratio that is 40:60 lactide:glycolide, a relaxin loaded at about 1%    by weight of the microparticles, and PVA in a concentration of about    0.5% by weight.-   129. The formulation of any of the preceding paragraphs; wherein    said formulation comprises microparticles suspended in a vehicle    solution.-   130. The formulation of any of the preceding paragraphs; wherein    said formulation comprises microparticles suspended in a sodium    chloride liquid solution.-   131. The formulation of any of the preceding paragraphs; wherein    said formulation comprises microparticles suspended in a sodium    chloride liquid solution; wherein said sodium chloride is 0.5-1.5    w/w%; or between 0.75-1.25 w/w%; or about 0.5 w/w%; or about 0.6    w/w%; or about 0.7 w/w%; or about 0.8 w/w%; or about 0.9 w/w%; or    about 1.0 w/w%; or about 1.1 w/w%; or about 1.2 w/w%; or about 1.3    w/w%; or about 1.4 w/w%; or about 1.5 w/w% of the liquid solution.-   132. The formulation of any of the preceding paragraphs; wherein    said formulation comprises microparticles suspended in a sodium    carboxymethylcellulose solution.-   133. The formulation of any of the preceding paragraphs; wherein    said formulation comprises microparticles suspended in a sodium    carboxymethylcellulose solution; wherein said sodium    carboxymethylcellulose solution is 0.1-1.0 w/w%; or between    0.25-0.75 w/w%; or about 0.1 w/w%; or about 0.2 w/w%; or about 0.3    w/w%; or about 0.4 w/w%; or about 0.5 w/w%; or about 0.6 w/w%; or    about 0.7 w/w%; or about 0.8 w/w%; or about 0.9 w/w%; or about 1.0    w/w% of the liquid solution.-   134. The formulation of any of the preceding paragraphs, wherein    said formulation is a sustained release formulation.-   135. The formulation of any of the preceding paragraphs, wherein    said formulation is a sustained release formulation wherein the    antifibrotic agent is released over an extended period of time.-   136. The formulation of any of the preceding paragraphs, wherein    said formulation is a sustained release formulation wherein the    antifibrotic agent is released over an extended period of least 1    day; or at least 2 days; or at least 3 days; or at least 4 days; or    at least 5 days; or at least 6 days; or at least 1 week; or at least    2 weeks; or at least 3 weeks; or at least 4 weeks; or at least 5    weeks, or at least 6 weeks; or at least 8 weeks; or at least 9    weeks; at least 10 weeks; or at least 12 weeks; or at least 15    weeks; or between 1-5 days; or between 2-5 days; or between 1-2    days; or between 2-3 days; or between 3-4 days; or between 4-5 days;    or between 3-10 days; or between 1-15 weeks; or between 2-10 weeks;    or between 4-8 weeks; or between 8-15 weeks; or about 1 day; or    about 2 days; or about 3 days; or about 4 days; or about 5 days; or    about 6 days; or about 1 week; or about 2 weeks; or about 3 weeks;    or about 4 weeks; or about 5 weeks; or about 6 weeks; or about 7    weeks; or about 8 weeks; or about 9 weeks; or about 10 weeks, or    more.-   137. A method, said method comprising identifying a subject    diagnosed with one or more diseases selected from the group of    diseases listed in Table 1 or Table 2 and administering a    formulation of any one of the preceding paragraphs to the subject.-   138. A method, said method comprising identifying a subject    diagnosed with Duchenne Muscular Dystrophy and administering to said    patient a composition or formulation of any of the preceding    paragraphs.-   139. A method, said method comprising identifying a subject    diagnosed with Becker Muscular Dystrophy and administering to said    patient a composition or formulation of any of the preceding    paragraphs.-   140. A method, said method comprising identifying a subject    diagnosed with Spinal Muscular Atrophy, Type I, and administering to    said patient a composition or formulation of any of the preceding    paragraphs.-   141. A method, said method comprising identifying a subject    diagnosed with Spinal Muscular Atrophy, Type II, and administering    to said patient a composition or formulation of any of the preceding    paragraphs.-   142. A method, said method comprising identifying a subject    diagnosed with Spinal Muscular Atrophy, Type III, and administering    to said patient a composition or formulation of any of the preceding    paragraphs.-   143. A method, said method comprising identifying a subject    diagnosed with Spinal Muscular Atrophy, Type IV, and administering    to said patient a composition or formulation of any of the preceding    paragraphs.-   144. A method, said method comprising identifying a subject    diagnosed with Cerebral Palsy, Stroke, Traumatic Brain Injury,    and/or peripheral nerve damage, and administering to said patient a    composition or formulation of any of the preceding paragraphs.-   145. A method, said method comprising identifying a subject    diagnosed with Arthrogryposis Multiplex Congenita and administering    to said patient a composition or formulation of any of the preceding    paragraphs.-   146. A method, said method comprising identifying a subject with    fibrosis of the humeroradial joint and administering to said patient    a composition or formulation of any of the preceding paragraphs.-   147. A method, said method comprising identifying a subject with    fibrosis of the humeroulnar joint and administering to said patient    a composition or formulation of any of the preceding paragraphs.-   148. A method, said method comprising identifying a subject with    fibrosis of the glenohumeral joint and administering to said patient    a composition or formulation of any of the preceding paragraphs.-   149. A method, said method comprising identifying a subject with    fibrosis of the tibiofemoral joint and administering to said patient    a composition or formulation of any of the preceding paragraphs.-   150. A method, said method comprising identifying a subject with    fibrosis of the acetabulofemoral joint and administering to said    patient a composition or formulation of any of the preceding    paragraphs.-   151. A method, said method comprising identifying a subject with    fibrosis of the talocrural joint and administering to said patient a    composition or formulation of any of the preceding paragraphs.-   152. A method, said method comprising identifying a subject with    fibrosis of the temporomandibular joint and administering to said    patient a composition or formulation of any of the preceding    paragraphs.-   153. A method, said method comprising identifying a subject with    fibrosis of the metacarpophalangeal joint and administering to said    patient a composition or formulation of any of the preceding    paragraphs.-   154. A method, said method comprising identifying a subject with    fibrosis of the metatarsophalangeal joint and administering to said    patient a composition or formulation of any of the preceding    paragraphs.-   155. A method, said method comprising identifying a subject with    fibrosis of the peri-articular musculature and administering to said    patient a composition or formulation of any of the preceding    paragraphs.-   156. A method, said method comprising identifying a subject with    cellulite and administering to said patient a composition or    formulation of any of the preceding paragraphs.-   157. A method, said method comprising identifying a subject with    interstitial lung disease and administering to said patient a    composition or formulation of any of the preceding paragraphs.-   158. A method, said method comprising administering, to any of the    preceding subjects, a composition or formulation of any of the    preceding paragraphs, via inhalation as an aerosol.-   159. A method, said method comprising administering, to any of the    preceding subjects, a composition or formulation of any of the    preceding paragraphs, via intra-articular injection.-   160. A method, said method comprising administering, to any of the    preceding subjects, a composition or formulation of any of the    preceding paragraphs, via intramuscular injection.-   161. A method, said method comprising administering, to any of the    preceding subjects, a composition or formulation of any of the    preceding paragraphs, via intradermal injection.-   162. A method, said method comprising administering, to any of the    preceding subjects, a composition or formulation of any of the    preceding paragraphs, via subcutaneous injection.-   163. A method, said method comprising administering, to any of the    preceding subjects, a composition or formulation of any of the    preceding paragraphs, via intracapsular injection.-   164. A method, said method comprising administering, to any of the    preceding subjects, a composition or formulation of any of the    preceding paragraphs, via pericapsular injection.-   165. A method, said method comprising administering, to any of the    preceding subjects, a composition or formulation of any of the    preceding paragraphs, via intraligamentous injection.-   166. A method, said method comprising administering, to any of the    preceding subjects, a composition or formulation of any of the    preceding paragraphs, via periligamentous injection.-   167. A method, said method comprising administering, to any of the    preceding subjects, a composition or formulation of any of the    preceding paragraphs, via intratendinous injection.-   168. A method, said method comprising administering, to any of the    preceding subjects, a composition or formulation of any of the    preceding paragraphs, via peritendinous injection.-   169. A method, said method comprising administering, to any of the    preceding subjects, a composition or formulation of any of the    preceding paragraphs, via intraosteotendinous injection.-   170. A method, said method comprising administering, to any of the    preceding subjects, a composition or formulation of any of the    preceding paragraphs, via periosteotendinous injection.-   171. A method, said method comprising administering, to a subject    diagnosed with Duchene’s muscular dystrophy, a composition or    formulation of any of the preceding paragraphs, via intramuscular    injection.-   172. A method, said method comprising administering, to a subject    diagnosed with Duchene’s muscular dystrophy, a composition or    formulation of any of the preceding paragraphs, via intraarticular    injection.-   173. A method, said method comprising administering, to a subject    diagnosed with Becker’s muscular dystrophy, a composition or    formulation of any of the preceding paragraphs, via intramuscular    injection.-   174. A method, said method comprising administering, to a subject    diagnosed with Becker’s muscular dystrophy, a composition or    formulation of any of the preceding paragraphs, via intraarticular    injection-   175. A method, said method comprising administering, to a subject    diagnosed with Spinal Muscular Dystrophy, a composition or    formulation of any of the preceding paragraphs, via intramuscular    injection.-   176. A method, said method comprising administering, to a subject    diagnosed with Spinal Muscular Dystrophy, a composition or    formulation of any of the preceding paragraphs, via intraarticular    injection.-   177. A method, said method comprising administering, to a subject    diagnosed with Arthrogryposis Multiplex Congenita, a composition or    formulation of any of the preceding paragraphs, via intramuscular    injection.-   178. A method, said method comprising administering, to a subject    diagnosed with Arthrogryposis Multiplex Congenita, a composition or    formulation of any of the preceding paragraphs, via intraarticular    injection.-   179. A method, said method comprising administering, to a subject    diagnosed with Cerebral Palsy, a composition or formulation of any    of the preceding paragraphs, via intramuscular injection.-   180. A method, said method comprising administering, to a subject    diagnosed with Cerebral Palsy, a composition or formulation of any    of the preceding paragraphs, via intraarticular injection.-   181. A method, said method comprising administering, to a subject    diagnosed with stroke, a composition or formulation of any of the    preceding paragraphs, via intramuscular injection.-   182. A method, said method comprising administering, to a subject    diagnosed with stroke, a composition or formulation of any of the    preceding paragraphs, via intraarticular injection.-   183. A method, said method comprising administering, to a subject    diagnosed with traumatic brain injury, a composition or formulation    of any of the preceding paragraphs, via intramuscular injection.-   184. A method, said method comprising administering, to a subject    diagnosed with traumatic brain injury, a composition or formulation    of any of the preceding paragraphs, via intraarticular injection.-   185. A method, said method comprising administering, to a subject    diagnosed with peripheral nerve damage, a composition or formulation    of any of the preceding paragraphs, via intramuscular injection.-   186. A method, said method comprising administering, to a subject    diagnosed with peripheral nerve damage, a composition or formulation    of any of the preceding paragraphs, via intraarticular injection.-   187. A method, said method comprising administering any of the    preceding paragraphs with sizes between 1um-10µm via inhalation as    an aerosol.-   188. A method, said method comprising administering any of the    preceding paragraphs with sizes between 20um-100µm via intramuscular    injection.-   189. A method, said method comprising administering any of the    preceding paragraphs with sizes between 5um-50µum via intraarticular    injection.-   190. A method, said method comprising administering, to a subject    diagnosed with interstitial lung disease any of the preceding    paragraphs via inhalation as an aerosol.-   191. A method, said method comprising administering, to a subject    diagnosed with interstitial lung disease any of the preceding    paragraphs via inhalation as an aerosol.-   192. A method, said method comprising administering, to a subject    diagnosed with interstitial lung disease any of the preceding    paragraphs, wherein the diameter of the microparticle is 1-10 µm,    via inhalation as an aerosol.-   193. A method, said method comprising administering, to a subject    diagnosed with Duchene’s Muscular Dystrophy any of the preceding    paragraphs, wherein the diameter of the microparticle is 10-30 µm,    via intraarticular injection.-   194. A method, said method comprising administering, to a subject    diagnosed with Duchene’s Muscular Dystrophy any of the preceding    paragraphs, wherein the diameter of the microparticle is 25-50 µm,    via intraarticular injection.-   195. A method, said method comprising administering, to a subject    diagnosed with Duchene’s Muscular Dystrophy any of the preceding    paragraphs, wherein the diameter of the microparticle is 10-30 µm,    via intramuscular injection.-   196. A method, said method comprising administering, to a subject    diagnosed with Duchene’s Muscular Dystrophy any of the preceding    paragraphs, wherein the diameter of the microparticle is 25-50 µm,    via intramuscular injection.-   197. A method, said method comprising administering, to a subject    diagnosed with Spinal Muscular Atrophy any of the preceding    paragraphs, wherein the diameter of the microparticle is 10-30 µm,    via intraarticular injection.-   198. A method, said method comprising administering, to a subject    diagnosed with Spinal Muscular Atrophy any of the preceding    paragraphs, wherein the diameter of the microparticle is 25-50 µm,    via intraarticular injection.-   199. A method, said method comprising administering, to a subject    diagnosed with Spinal Muscular Atrophy any of the preceding    paragraphs, wherein the diameter of the microparticle is 10-30 µm,    via intramuscular injection.-   200. A method, said method comprising administering, to a subject    diagnosed with Spinal Muscular Atrophy any of the preceding    paragraphs, wherein the diameter of the microparticle is 25-50 µm,    via intramuscular injection.-   201. A method, said method comprising administering, to a subject    diagnosed with joint arthrofibrosis any of the preceding paragraphs,    wherein the diameter of the microparticle is 10-30 µm, via    intraarticular injection.-   202. A method, said method comprising administering, to a subject    diagnosed with joint arthrofibrosis any of the preceding paragraphs,    wherein the diameter of the microparticle is 25-50 µm, via    intraarticular injection.-   203. A method or formulation of any of the preceding paragraphs,    wherein the formulation is delivered via inhalation as an aerosol.-   204. A method or formulation of any of the preceding paragraphs,    wherein the formulation is delivered via intra-articular injection.-   205. A method or formulation of any of the preceding paragraphs,    wherein the formulation is delivered via intramuscular injection.-   206. The method or formulation of any one of the preceding    paragraphs wherein, the formulation is administered to the subject    such that the antifibrotic agent (e.g., a relaxin) is administered    to a subject at a dose between 1-2000 µg/kg body weight; or between    10-100 µg/kg body weight; or between 100-200 µg/kg body weight; or    between 200-500 µg/kg body weight; or between 500-1000 µg/kg body    weight; or 25-75 µg/kg body weight; or 30-70 µg/kg body weight; or    40-60 µg/kg body weight; or between 1-10 µg/kg body weight; or    between 1-5 µg/kg body weight; or between 4-8 µg/kg body weight; or    about 2 µg/kg body weight; or about 5 µg/kg body weight; or about 10    µg/kg body weight; or about 20 µg/kg body weight; or about 25 µg/kg    body weight; or about 30 µg/kg body weight; or about 35 µg/kg body    weight; or about 40 µg/kg body weight; or about 45 µg/kg body    weight; or about 50 µg/kg body weight; or about 55 µg/kg body    weight; or about 60 µg/kg body weight; or about 65 µg/kg body    weight; or about 70 µg/kg body weight; or about 75 µg/kg body    weight; or about 100 µg/kg body weight; or about 200 µg/kg body    weight; or about 500 µg/kg body weight.-   207. The method or formulation of any one of the preceding    paragraphs, wherein the formulation is administered one time.-   208. The method or formulation of any one of the preceding    paragraphs, wherein the formulation is administered more than one    time.-   209. The method or formulation of any one of the preceding    paragraphs, wherein the formulation is administered once per week.-   210. The method or formulation of any one of the preceding    paragraphs, wherein the formulation is administered once every 2    weeks.-   211. The method or formulation of any one of the preceding    paragraphs, wherein the formulation is administered once every 3    weeks.-   212. The method or formulation of any one of the preceding    paragraphs, wherein the formulation is administered once every 4    weeks.-   213. The method or formulation of any one of the preceding    paragraphs, wherein the formulation is administered once every 6    weeks.-   214. The method or formulation of any one of the preceding    paragraphs, wherein the formulation is administered once every 8    weeks.-   215. The method or formulation of any one of the preceding    paragraphs, wherein the formulation is administered once every 10    weeks.-   216. The method or formulation of any one of the preceding    paragraphs, wherein the formulation is administered once every 12    weeks.-   217. The method or formulation of any one of the preceding    paragraphs, wherein the formulation is administered once every 16    weeks.-   218. The method or formulation of any one of the preceding    paragraphs, wherein the formulation is administered once every 20    weeks.-   219. The method or formulation of any one of the preceding    paragraphs, wherein the formulation is administered once every 24-26    weeks.-   220. The method or formulation of any one of the preceding    paragraphs, wherein the formulation is administered once every 8    months.-   221. The method or formulation of any one of the preceding    paragraphs, wherein the formulation is administered once every 9    months.-   222. The method or formulation of any one of the preceding    paragraphs, wherein the formulation is administered once every 12    months.-   223. The method or formulation of any one of the preceding    paragraphs, wherein the antifibrotic agent is released from the    microparticle with a linear release profile.-   224. The method or formulation of any one of the preceding    paragraphs, wherein the antifibrotic agent is released from the    microparticle with a non-linear release profile.-   225. The method or formulation of any one of the preceding    paragraphs, wherein the antifibrotic agent is released from the    microparticle with an initial bolus in its release profile.-   226. The method or formulation of any one of the preceding    paragraphs, wherein the antifibrotic agent is released from the    microparticle without an initial bolus in its release profile.-   227. The method or formulation of any one of the preceding    paragraphs, wherein the antifibrotic agent is released from the    microparticle with a burst-release in its release profile after the    first several days.-   228. The method or formulation of any one of the preceding    paragraphs, wherein the antifibrotic agent is released from the    microparticle with no burst-release in its release profile.

Some embodiments of the technology described herein can be definedaccording to any of the following additional numbered paragraphs:

-   1. A method of treating a fibrotic disease, the method comprising    administering to a subject in need thereof an agent that binds a    relaxin family peptide receptor.-   2. The method of any preceding paragraphs, wherein the relaxin    family peptide receptor is RXFP1, RXFP2, RXFP3, or RXFP4.-   3. The method of any preceding paragraphs, wherein the agent is a    native ligand of the receptor.-   4. The method of any preceding paragraphs, wherein the native ligand    is Relaxin-2 or a Relaxin-2 variant.-   5. The method of any preceding paragraphs, wherein the Relaxin-2    variant is at least 85%, at least 90%, at least 95% or at least 99%    similar to native Relaxin-2.-   6. The method of any preceding paragraphs, wherein the agent is    recombinantly produced in a bacterial, mammalian or yeast host cell.-   7. The method of any preceding paragraphs, wherein the agent is    fully or partially chemically synthesized.-   8. The method of any preceding paragraphs, wherein the agent is    conjugated to a targeting moiety.-   9. The method of any preceding paragraphs, wherein the targeting    moiety is selected from the group consisting of a single-domain    camelid antibody fragment, a peptide sequence, polynucleotide, or a    small molecule, or a small molecule allosteric modulator.-   10. The method of any preceding paragraphs, wherein the agent is    comprised in a depot.-   11. The method of any preceding paragraphs, wherein the depot has at    least one of    -   a. a volume of 0.1 um³;    -   b. is comprised of one or more polymer; or    -   c. is comprised of one or more self-assembled small molecule.-   12. The method of any preceding paragraphs, wherein the depot has a    diameter of 1-100 µm.-   13. The method of any preceding paragraphs, wherein the depot is    comprised of a hydrogel comprised of low molecular weight gelators.-   14. The method of any preceding paragraphs, wherein depot is    comprised of poly(lactic-co-glycolic acid).-   15. The method any preceding paragraphs, wherein the depot is    comprised of a crosslinked hydrogel comprised of polyethyelene    glycol.-   16. The method of any preceding paragraphs, wherein the depot is    comprised of a self-assembled amphiphilic hydrogel comprised of    amphiphilic small molecules.-   17. The method of any preceding paragraphs, wherein the fibrotic    disease is selected from the group consisting of stiffened fibrotic    joint capsules, lung fibrosis (i.e. idiopathic pulmonary fibrosis,    cystic fibrosis, hypertension), liver fibrosis (i.e. hepatitis B or    C, long-term alcohol abuse, non-alcoholic steatohepatitis,    non-alcoholic fatty liver disease, Cholestasis, autoimmune hepatitis    cirrhosis), kidney fibrosis (i.e. chronic kidney disease, end-stage    renal disease, renal interstitial fibrosis), heart disease (i.e.    heart failure, myocardial infarction, aortic stenosis, hypertrophic    cardiomyopathy), intestinal disease (i.e. Crohn’s disease,    inflammatory bowel disease, enteropathies, and other intestinal    fibrosis), skin conditions (i.e. scleroderma, keloids, hypertrophic    scars, cellulite), urogenital and gynecological conditions    (Peyronie’s disease, uterine fibroids) and ocular diseases (i.e.    Congenital Fibrosis of the Extraocular Muscles, subretinal fibrosis,    epiretinal fibrosis, corneal fibrosis).-   18. The method of any preceding paragraphs, wherein the agent is    administered locally to a target organ.-   19. The method of any preceding paragraphs, wherein the agent is    administered systemically.-   20. The method of any preceding paragraphs, wherein the agent is    administered via intraarticular, periarticular, intracapsular,    pericapsular, intraligamentous, periligamentous, intratendinous,    peritendinous, intraosteotendinous, or periosteotendinous injection,    intramuscularly, subcutaneously, intradermally, intranasally,    orally, transcutaneously, mucosally, transcutaneously, or by    pulmonary inhalation.-   21. The method of any preceding paragraphs, wherein administration    is under fluoroscopic or ultrasound guidance.-   22. The method of any preceding paragraphs, wherein the target organ    is a lung, kidney, liver, heart, skin or eye.-   23. The method of any preceding paragraphs, wherein the subject is    further administered at least a second therapeutic.-   24. The method of any preceding paragraphs, wherein the at least a    second therapeutic is physical therapy.-   25. The method of any preceding paragraphs, wherein the at least a    second therapeutic is a surgery.-   26. The method of any preceding paragraphs, comprising a step, prior    to administering, of diagnosing a subject as having a fibrotic    disease.-   27. The method of any preceding paragraphs, comprising a step, prior    to administering, of receiving a results of an assay that diagnoses    a subject as having a fibrotic disease.-   28. A method of treating a fibrotic disease, the method comprising    administering to a subject in need thereof an agent that binds a    relaxin family peptide receptor, wherein the agent is comprised in a    depot for sustained release.-   29. A composition comprising an agent that binds a relaxin family    peptide receptor.-   30. The composition of any preceding paragraphs, wherein the relaxin    family peptide receptor is RXFP1, RXFP2, RXFP3, or RXFP4.-   31. The composition of any preceding paragraphs, wherein the agent    is a native ligand of the receptor.-   32. The composition of any preceding paragraphs, wherein the native    ligand is Relaxin-2 or a Relaxin-2 variant.-   33. The composition of any preceding paragraphs, wherein the    Relaxin-2 variant is at least 85%, at least 90%, at least 95% or at    least 99% similar to native Relaxin-2.-   34. The composition of any preceding paragraphs, wherein the agent    is recombinantly produced in a bacterial, mammalian or yeast host    cell.-   35. The composition of any preceding paragraphs, wherein the agent    is fully or partially chemically synthesized.-   36. The composition of any preceding paragraphs, wherein the agent    is conjugated to a targeting moiety.-   37. The composition of any preceding paragraphs, wherein the    targeting moiety is selected from the group consisting of a    single-domain camelid antibody fragment, a peptide sequence,    polynucleotide, or a small molecule, or a small molecule allosteric    modulator.-   38. The composition of any preceding paragraphs, wherein the agent    is comprised in a depot.-   39. The composition of any preceding paragraphs, wherein the depot    has at least one of    -   a. a volume of 0.1 um³;    -   b. is comprised of one or more polymer; or    -   c. is comprised of one or more self-assembled small molecule.-   40. The composition of any preceding paragraphs, wherein the depot    has a diameter of 1-100 µm.-   41. The composition of any preceding paragraphs, wherein the depot    is comprised of a hydrogel comprised of low molecular weight    gelators.-   42. The composition of any preceding paragraphs, wherein depot is    comprised of poly(lactic-co-glycolic acid).-   43. The composition of any preceding paragraphs, wherein the depot    is comprised of a crosslinked hydrogel comprised of polyethyelene    glycol.-   44. The composition of any preceding paragraphs, wherein the depot    is comprised of a self-assembled amphiphilic hydrogel comprised of    amphiphilic small molecules-   45. Use of a composition of any preceding paragraphs, for treatment    of a fibrotic disease.

EXAMPLES

The present invention will be further described in the followingexamples, which do not limit the scope of the present invention.

Example 1. Synthesis, Characterization, and Evaluation ofRelaxin-2-Loaded Microparticles

Relaxin-2 loaded PLGA microparticles (RMPs) were prepared based on thewater/oil/water (w/o/w) method as described by Igartua, M., et. al.,International Journal of Pharmaceutics, 1998, 169(1): 45-54 at 1.27 x10-3 wt % loaded relaxin as determined by ELISA analysis. Significantoptimization and tailoring of the method was required to obtain theparticle. Amounts, time, temperature, mixing speed, polymer LA:GAcontent were all varied to identify a unique non-obvious set ofconditions to prepare the polymer. An ELISA analysis also revealed anencapsulation efficiency of 90-95%. Dynamic light scattering (DLS)showed the particles to be 7.65 µm in diameter with a smallpolydispersity and spherical shape by SEM. Relaxin-2 in the relaxin-2microparticles maintained its native secondary and tertiary structurethrough the w/o/w double-emulsion process. Circular dichroism spectrashowed no change between freshly prepared and emulsified relaxin-2(FIG.2 , left). Additionally, relaxin-2 was stable at 37° C. over 2 months(FIG. 2 , right). The relaxin-2 PLGA microparticles were prepared withbatch-to-batch consistency and were resuspended in PEG-600 to reduceaggregation and flocculation, as well as to ease administration througha 23G needle.

Relaxin-2 microparticles are prepared by spray drying. A rotary wheelatomizer spinning at 15,000 RPM with peripheral exit speeds of 250 m/secand an air flow rate of 40 m/sec produces 35 µm relaxin-2microparticles. The feed solution comprises an emulsion of relaxin-2 (1mg/ml in H₂O) and PL:GA (molar ratio 45:65 lactide:glycolide, M.W.50,000-75,000 daltons, carboxylic acid terminated, 50 mg/ml in methylenechloride).

Relaxin-2 microparticles are prepared by solvent extraction. An emulsionof relaxin-2 (0.5 mg/ml) and PL:GA (molar ratio 50:50 lactide:glycolide,M.W. 70,000-90,000 daltons, ester terminated, 50 mg/ml) is formed inethyl acetate. 1% by volume poly-vinyl alcohol (M.W. 30,000 dalton) isadded to the rapidly mixing emulsion and then the remaining solvent isremoved by evaporation. Prior to lyophilization, the relaxin-2micropartilces are 48 µm in diameter.

The release of relaxin-2 from the microparticles was quantified via arelaxin-2 ELISA. The concentration of relaxin-2 in a reservoir ofbiologically relevant buffer meant to mimic synovial fluid (Dublecco’sModified Eagle Media + 20% heat inactivated fetal bovine serum + 25µg/ml porcine esterase + 1% penicillin streptomycin) was detereminedevery three days over the course of 60 days. After 28 days, 85 % of theinitial encapsulated relaxin-2 was released from the particles (FIG. 3 ,top). Release occurs at a semi-linear rate for the first four weeks(FIG. 3 , top). SEM image analysis of relaxin-2 microparticles atvarious time points showed that into the 3rd week, the relaxinmicroparticles degrade via bulk erosion. At the 5^(th) and 6^(th) week,the relaxin-2 microparticles displayed near complete degradation withhigh porosity and me to degrade as the smooth surface is lost andbecomes wrinkled. By the fourth week, the RMPs are significantlydegraded as indicated by their substantial increase in porosity comparedto when initially prepared (FIG. 3 , bottom).

Human fibroblast-like synoviocytes (HFLS) treated with relaxin-2microparticles in transwells allow for evaluation of the release andefficacy of released relaxin-2 at various time points. Relaxin-2released from relaxin-2 microparticles demonstrated antifibroticefficacy for up to four weeks as indicated by the downregulation ofcollagen I expression in the presence of 5 ng/mL TGFβ-1 (FIG. 4 , top).In the presence of relaxin-2, collagen I is expressed at 20 to 30 % ofthe basal level at weeks 1, 3, and 4. The 2-week time point displayed 50% downregulation of collagen expression(FIG. 4 , bottom).

The synovial joint space consists of two major cell types: synovialfibroblasts and macrophages. The in vitro tolerability of RAW 267.4murine macrophages to relaxin-2 microparticles was evaluated. After a24-hour treatment, macrophages internalized fluorescently labelledcontrol microparticles with average diameter of 4.5 µm (FIG. 5 , left).After 24-hour treatment, macrophages did not internalize fluorescentlylabelled control microparticles, with average diameter of 7.6 µm (FIG. 5, right).

Example 2. Formation and Characterization of Amphiphilic Hydrogel Depotsfor the Sustained Release of Relaxin-2

Two amphiphilic hydrogels were synthesized for the physical entrapmentof relaxin-2 in order to control its sustained release (FIG. 6 ). Theseamphiphilic molecules melted at 95 oC and congealed when slowly cooledback to room temperature strictly from non-covalent interactions. Thealkyl chain of 1 and alkyl fluoride chain of 2 allow for hydrophobicinteractions between each molecule, while the trizol and thymidinemoieties allow for pi-pi stacking. Lastly, the deoxyribose and glucosegroups facilitate hydrogen bonding (Godeau, G., et al., TetrahedronLetters 2010, 51: 1012-1015; Ramen, F.A. et al., Biomaterials. 2017,145: 72-80). Together, these amphiphilic molecules create unique andstable supramolecular assemblies that are stable at 37 oC for at leasttwo weeks. This stability feature is another novel finding and benefitto the use of the hydrogels. Three gels of each 1 and 2 were createdwith bovine serum albumin and relaxin-2 at different densities bydissolving each molecule in a solution of phosphate buffered saline withprotein and heating to 95 oC for 30 minutes. Relaxin-2 maintains itsfolded 3D structure by circular dichroism spectroscopy after treatmentat this temperature for at least one hour (FIG. 7 , left). After coolingdown to room temperature, relaxin-2-loaded gels were fully congealed(FIG. 7 , right).

The rheological properties of these supramolecular hydrogels loaded withrelaxin-2 were assessed. The storage (G′) and loss (G″) moduli weredetermined from a frequency sweep from 1 to 100 rad/s. In this range,all gels maintained a greater storage modulus than loss modulus (G′ >G″) indicating that all gels behave more as an elastic solid thanviscous liquid in this frequency regime (FIG. 8 , left, FIG. 9 , left).A strain sweep of gel 1 at various wt % reveals the linear viscoelasticregions (LVERs) between 2.2% strain to 6.4% strain from high to low wt %gels, respectively (FIG. 8 , right). The LVERs are inversely related tothe gel wt %. Alternatively, in gel 2 at various wt %, this relationshipis lost (FIG. 9 , right). The 5 wt % gel shows the narrowest LVER,ending at 9 % strain. The 1.5 wt % gel shows the largest LVER ending at30 %, and the 1 wt % gel is intermediate between the two with a LVERending at 20 % strain (FIG. 9 , right).

The release profile of 1 and 2 wt % in 1 and 1 and 1.5 wt % in 2 wereassessed over the course of 8 days at 37 oC in a reservoir of phosphatebuffered saline. In this time frame, both gels created from molecule 2released 90 and 84 % of the total loaded relaxin-2 (FIG. 10 ). The gelsprepared from compound 1 exhibited a significantly slower releasepattern than those of compound 2. These gels release only 15 and 27 % ofthe total loaded relaxin-2 (FIG. 10 ). The gels made from compound 1show a proportionality between release time and wt %, whereas this trendis absent in gels made from compound 2. Over the course of 8 weeks, allgels remained intact with some swelling observed. There were no apparentsigns of the material degrading. The understanding for why these twogels release relaxin at different rates is unknown and as such points tothis remarkable finding.

Example 3: Development of a Shoulder Contracture Model in Rats

The purpose of this experiment, which is described in the publication byVilla-Camacho et al., Journal of Shoulder and Elbow Surgery, 2015,24(11): 1809-16, was to investigate the effects of extraarticular,internal fixation of the glenohumeral joint on shoulder kinetics andkinematics in an in vivo animal model of shoulder contracture. It wasexpected that extraarticular, internal fixation of the shoulder in ratswould result in a subsequent decrease in rotational ROM and an increasein joint stiffness, which would persist for at least 8 weeks.

The study was approved by the Institutional Animal Care and UseCommittee, and 10 Sprague-Dawley rats (250-300 g, Charles RiverLaboratories, Wilmington, MA, USA) were used in the study. For eachanimal, torque was measured per degree, on the intact left shoulder as afunction of rotation angle between 80° of internal rotation (negativevalues by convention) and 60° of external rotation (positive values byconvention) prior to any surgical intervention (baseline). Rotation wasconfined within boundaries that were observed to elicit minimal scapularrecruitment, as confirmed by fluoroscopy. Therefore, torque values at60° external rotation (rOUT) and 80° of internal rotation (τINT) wererecorded for each animal.

The left forelimb of each animal was immobilized using extraarticularinternal fixation. Under isoflurane anesthesia, a longitudinal skinincision was made perpendicular to the scapular spine. Two No. 2-0braided polyester sutures (Ethibond Excel, Ethicon - San Lorenzo, PR,USA) were passed between the medial border of the scapula and thehumeral shaft and tightened to immobilize the shoulder joint (FIG. 1 ,panel A). Muscular structures were not manipulated during surgery, andthe animals were allowed normal activity in their cages immediatelyafter the procedure.

After 8 weeks of immobilization, the restraining sutures were removed,and the 10 animals were divided into two groups to evaluate changes inROM (ROM group, n = 5) and joint stiffness (stiffness group, n = 5). Inthe ROM group, changes in kinematics were longitudinally quantified inthe follow-up period by measuring the ROM achieved with the τOUT andτINT measured at baseline. This was conducted to evaluate whetherimmobilization mediated a significant reduction in ROM. In the stiffnessgroup, joint kinetics were examined by measuring the differences in τOUTand τINT needed to achieve the original 80° of internal rotation and 60°of external rotation, respectively. Measurements for both groups weretaken immediately after suture removal (day 0 of follow-up) and atregular intervals thereafter (twice a week until less than 10% changewas observed in three consecutive time points, at which point,measurement frequency was reduced to once a week). The baselinemeasurements for each group were used as internal controls to reduce thetotal number of animals necessary for the study. The use of internalcontrols also increased internal validity and statistical power as therewas a high inter-specimen variation, of both ROM and measured torques,even when using the contralateral shoulder of the same animal. Finally,a pilot study demonstrated that intra-specimen measurements were highlyreproducible and remained stable during an 8-week period.

ROM and torque measurements were performed under general anesthesiausing a device that consisted of a sensor assembly, a rotating axle, andan arm clamp. The sensor assembly contained an orientation sensor(3DM-GX3-15, MicroStrain - Williston, VT), as well as a reaction torquesensor (TFF400, Futek - Irvine, CA) secured to the axle such that thesensing axis was collinear with the center of rotation. The forelimb wassecured at 3 points (wrist, elbow, and arm), ensuring that the sensingaxis was aligned with the long axis of the humerus. Rotation of thesensor assembly resulted in direct internal humeral rotation andexternal humeral rotation within the glenohumeral joint.

To reproducibly capture ROM and torque, passive limb rotation wasperformed by a stepper motor controlled with a microcontroller (UNO R3,Arduino - Torino, Italy). The system utilized inputs from the reactiontorque sensor or the orientation sensor to start and end the dynamicmeasurement of ROM and torque. In the ROM group, pre-set programmabletorque values, specific for each animal and measured at baseline (τOUTand τINT), were used as input variables in order to detect changes inrotation ROM with 0.2° resolution. In the stiffness group, pre-setprogrammable rotation angles (60° external rotation, 80° internalrotation) were used as input to measure changes in torque at aresolution of 0.01 N/mm. The microcontroller was directed by a computerusing MATLAB 7.13.0.564 (MathWorks Inc - Natick, MA, USA).

In the ROM group, mean ROM values were compared at three different timepoints (baseline, immediately after suture removal, and at 8 weeks offollow-up) by repeated-measures analysis of variance. In the stiffnessgroup, two different metrics were used for comparison: 1) the differencein torque required to achieve full ROM, and 2) stiffness, estimated fromthe area under the rotation angle-torque curve. A value of P < 0.05 wasconsidered statistically significant for both groups. The ROM temporalbehavior in the follow-up period was determined. Immediately aftersuture removal, there was a 63% decrease in total ROM compared withbaseline (51°± 10° vs. 136° ± 0°; P < 0.001). Similarly, total torqueincreased 13.4 N.mm compared with baseline (22.6 ± 5.9 N.mm vs. 9.2 ±2.6 N.mm; P = 0.002). Residual total ROM restrictions and an increasedtorque in internal rotation were still evident at 8 weeks of follow-up(113° ± 8° vs. 137° ± 0°, P < 0.001 and 3.5 ± 0.4 N.mm vs. 2.7 ± 0.7N.mm, P = 0.036).

The kinetic and kinematic changes were not transitory. At 8 weeksfollow-up, both the reduction in ROM and the increase in joint stiffnesswere significant. While no studies have evaluated the naturalprogression and temporal behavior of this shoulder contracture model, itis expected that joint residual changes present after 8 weeks into thepost-immobilization period are likely permanent (Trudel G. et al.,Journal of Applied Physiology (Bethesda, Md : 1985), 2014,117(7):730-7). The results presented in Example 4 indicate that ashoulder contracture model in rats may be used to evaluate therapeuticinterventions to treat shoulder contracture.

The above described findings were subsequently validated by Kim et al.,who independently reported a similar model of shoulder contracture (Kimet al., J. Orthop. Surg. Res. 2016; 11(1): 160). A series of microscopicimages of the axillary recess of the glenohumeral joint was similar tothat as seen in Kim et al., taken over 6 weeks. The Masson’s trichromestain was utilized to identify fibrosis (red). Histologic evidence ofcontracture development at 3 days and 6 weeks is observed. Fibrosis andinflammation occurred early and persisted during immobilization, andnotably, the infiltration of inflammatory cells, capsular thickening,and angiogenesis within capsular tissue was apparent as early as 3 days.While the acute inflammatory response lessened by week 6, capsularthickening and fibrotic structures still remained, closely mimickingfindings from other studies (Trudel et al., J. Appl. Physiol. (1985),2014, 117(7):730-7). This model of lasting reduced ROM and increasedstiffness allows for the comprehensive evaluation of current andpotential therapeutic interventions for shoulder contracture.

Example 4. Use of a Relaxin-2 Microparticle to Treat a Stiffened Jointin a Murine Model

This study investigated the effects of recombinant human relaxin-2 onthe kinetics and kinematics of the glenohumeral joint in an animal modelof shoulder contracture, as described in Villa-Camacho et al., Journalof Shoulder and Elbow Surgery, 2015, 24(11): 1809-16. It was expectedthat rats treated with multiple intraarticular (mIA) injections ofrelaxin-2 would exhibit a more rapid recovery of range of motion (ROM)throughout an 8 week monitoring period than untreated controls. For eachanimal, torque was measured per degree, on the intact left shoulder as afunction of rotation angle between 100° of internal rotation and 60° ofexternal rotation prior to any surgical intervention. After 8 weeks ofimmobilization, ROM was measured and rats were injected with relaxin-2at 2 ug/kg in phosphate buffered saline (i.e., vehicle group) directlyinto the synovial joint space of the stiff shoulder under fluoroscopicguidance (Day 0). Rats received a total of 5 injections of relaxin-2 at2 ug/kg administered every other day for 10 days for a total of 10 ug/kgadministered per rat (i.e., MIA group). Rats were injected on Day 0 witha single dose of PLGA microparticles encapsulating approximately 10ug/kg relaxin-2 (i.e., RMP group). As an additional control a separateset of animals receive no shoulder contracture and receive a total of 5injections of relaxin-2 at 2 ug/kg every other day for 10 days for atotal of 10 ug/kg administered per rat (i.e.,Healthy + MIA group).Changes in kinematics were longitudinally quantified starting at Day 0and in the follow-up period by measuring the ROM achieved with the τOUTand τINT measured at baseline. ROM measurements were taken immediatelyafter suture removal (day 0 of follow-up) and at regular intervalsthereafter (twice a week until less than 10% change was observed inthree consecutive time points, at which point, measurement frequency wasreduced to once a week) for an 8-week follow-up period. The baselinemeasurements for each group functioned as internal controls in additionto the same ROM measurements on the contralateral uninjured shoulder.Rats receiving mIA of relaxin-2 or relaxin loaded in the microparticle(RMP) demonstrated restoration of internal ROM to baseline measurementsafter the 8-week follow-up period (FIG. 11 ). Using this model, asingular articular injection or relaxin or an intravenous injection ofrelaxin-2 at a much greater dose shows a similarly constricted internalROM compared with the untreated control group. Histological analysis ofthe shoulder joint demonstrated that immobilized shoulders of untreatedrats has reduced delineated separation between the capsule and thearticular surface on the humeral head, fibroblast infiltration andcapsular adhesions, while immobilized shoulders of rats that receivedmIA of relaxin-2 (Blessing, W.A., et al., Proc. Natl. Acad. Sci. 2019,116: 12183 - 12192) or relaxin loaded microparticles lacked any apparentadhesions and the synovial membrane and the articular cartilage surfacesremained separated from one another with proper cellular organization,and displayed reduced foreign body response compared to vehiclemicroparticle control (FIG. 12 ).

Using the contracture model model above, animals at day 0 receivevehicle (empty microparticle) or relaxin loaded microparticles (i.e.,SR-RLX) and were followed for 8 weeks. FIG. 13 shows range of motionrecovery of the shoulder of rats receiving a single intra-articularinjection or relaxin-2 loaded microparticles was similar to that of ratsreceving 5 intra-articular inections of relaxin-2. Control unloadedvehicle microparticles and control saline injections demonstratedsimilar ROM recovery. This recovery was less than that of the relaxin-2treatment groups data for total, internal and external angles. There isa statistically significant difference between the total range of motionin rats treated with relaxin loaded depots (n=7) and unloaded vehiclecontrols (n=9) at the 14 and 16 day time point (p<0.01). This data arein agreement with the results from FIG. 11 .

Example 5. Use of Relaxin-2 Loaded Microparticles to Treat a StiffenedJoint in a Human Subject

Relaxin-2 microparticles, 35 µm in diameter, comprised of PLGA (molarratio 45:65 lactide:glycolide, M.W. 50,000-75,000 daltons, carboxylicacid terminated), and loaded at 1% relaxin weight/weight, areadministered to a patient diagnosed with shoulder adhesive capsulitis.Prior to injection, relaxin-2 microparticles are resuspended in asterile, isotonic carboxymethylcellulose diluent to a total volume suchthat the final dose is 50 µg/kg body weight. Administration is in theform of 1ml intraarticular injection using a 23G needle. Followinginjection, the patient is monitored for changes in joint range ofmotion, (e.g. internal rotation, external rotation, pronation,supination, flexion, extension, abduction, and adduction) patientreported pain, mobility, patient reported autonomy, and patient reportedquality of life.

Example 6. Use of Relaxin-2 Loaded Microparticles to Treat StiffenedJoints Secondary to a NMD

Relaxin microparticles, 35 µm in diameter, comprised of PLGA (molarratio 50:50 lactide:glycolide, M.W. 70,000-90,000 daltons, esterterminated) and PVA (M.W. 30,000 daltons), and loaded at 0.5% relaxinweight/weight, are administered to a patient diagnosed with Duchene’sMuscular Dystrophy presenting with contracture of the acetabulofemoraljoint. Prior to injection, relaxin microparticles are resuspended insterile saline-buffer-based diluent to a total volume such that thefinal dose is 20 ug/kg body weight. Administration is in the form of 1ml intraarticular injection using a 21G needle. Subsequent injections of1 ml at 10 ug/kg are performed every 14 days for 70 days following theinitial injection. Following in initial injection, during repeateddosages, and after completion of dose regime, the patient is monitoredfor changes in joint range of motion, (e.g. internal rotation, externalrotation, pronation, supination, flexion, extension, abduction, andadduction) patient reported pain, mobility, patient reported autonomy,and patient reported quality of life.

Example 7. Use of Relaxin-2 Loaded Microparticles to Treat PulmonaryFibrosis

Relaxin microparticles, 3.1 µm in diameter, comprised of PLGA, andloaded at 0.5% relaxin weight/weight, and administered to a patientdiagnosed with idiopathic pulmonary fibrosis presenting with reducedforced vital capacity and a dry cough. Prior to administration, relaxinmicroparticles are gentle agitated to deagglomerate any dry powderclumps that formed during storage. Using a dry powder metered doseinhaler, that patient is given an inhaled dose equivalent to 25 ug/kg.Following administration, the patient is monitored for decreases inpathological hallmarks of fibrosis via CT scan, as well as for increasedforced vital capacity, and decrease in respiratory distress symptoms

Example 8. Use of Relaxin-2 for the Treatment of Fibrosis

The present invention provides compositions of matter and methods fortreating fibrotic diseases including stiffened fibrotic joint capsules,lung fibrosis (i.e. idiopathic pulmonary fibrosis, cystic fibrosis,hypertension), liver fibrosis (i.e. hepatitis B or C, long-term alcoholabuse, non-alcoholic steatohepatitis, non-alcoholic fatty liver disease,Cholestasis, autoimmune hepatitis cirrhosis), kidney fibrosis (i.e.chronic kidney disease, end-stage renal disease, renal interstitialfibrosis), heart disease (i.e. heart failure, myocardial infarction,aortic stenosis, hypertrophic cardiomyopathy), intestinal disease (i.e.Crohn’s disease, inflammatory bowel disease, enteropathies, and otherintestinal fibrosis), skin conditions (i.e. scleroderma, keloids,hypertrophic scars, cellulite), urogenital and gynecological conditions(Peyronie’s disease, uterine fibroids) and ocular diseases (i.e.Congenital Fibrosis of the Extraocular Muscles, subretinal fibrosis,epiretinal fibrosis, corneal fibrosis) in a subject by administering adepot containing a binding agent for relaxin family peptide receptors(RXFP1, RXFP2, RXFP3, RXFP4). The agent will be the native ligand of thereceptor, relaxin-2, a relaxin-2 variant, relaxin-2 chemicallyconjugated to a targeting agent, including a single-domain camelidantibody fragment, a peptide sequence, polynucleotide, or a smallmolecule, or a small molecule allosteric modulator The depot is anobject with a volume of at least 0.1 µm3 and is comprised of one or morepolymers or self-assembled small molecules that delivers the minimallyeffective clinical dose over several weeks to several months. Therelaxin loaded depot may be administered intravenously, intramuscularly,subcutaneously, intradermally, intranasally, orally, transcutaneously,mucosally, intraarticularly, periarticularly, intracapsularly,pericapsularly, intratendinously, peritendinously, intraligamentously,periligamentously, by pulmonary inhalation or by ocular specific routesof administration as a sustained release formulation and may be providedas a single injection or a series of injection.

LISTING OF SEQUENCES

SEQ ID NO: 1 >gi116497221HgbAA126416.1 Relaxin 2 [Homo sapiens]MPRLFFFHLLGVCLLLNQFSRAVADSWMEEVIKLCGRELVRAQIAICGMSTWSKRSLSQEDAPQTPRPVAEIVPSFINKDTETINMMSEFVANLPQELKLTLSEMQPALPQLQQHVPVLKDSSLLFEEFKKLIRNRQSEAADSSPSELKYLGLDTHSRKKRQLYSALANKCCHVGCTKRSLARFC

SEQ ID NO: 2 >gi1116496899IgbIAAI26420.1I Relaxin 2 [Homo sapiens]MPRLFFFHLLGVCLLLNQFSRAVADSWMEEVIKLCGRELVRAQIAICGMSTWSKRSLSQEDAPQTPRPVAEIVPSFINKDIETINMMSEFVANLPQELKLILSEMQPALPQLQQHVPVLKDSSLLFEEFKKLIRNRQSEAADSSPSELKYLGLDTHSRKKRQLYSALANKCCHVGCTKRSLARFC

SEQ ID NO: 3 >gi1313884020IgbIADR83496.1I relaxin 2, partial [synthetic construct]MPRLFFFHLLGVCLLLNQFSRAVADSWMEEVIKLCGRELVRAQIAICGMSTWSKRSLSQEDAPQTPRPVAEIVPSFINKDIETINMMSEFVANLPQELKLILSEMQPALPQLQQHVPVLKDSSLLFEEFKKLIRNRQSEAADSSPSELKYLGLDTHSRKKRQLYSALANKCCHVGCTKRSLARFC

SEQ ID NO: 4 >gi113543609IgbIAAH05956.1I Relaxin 1 [Homo sapiens]MPRLFLFHLLEFCLLLNQFSRAVAAKWKDDVIKLCGRELVRAQIAICGMSTWSKRSLSQEDAPQTPRPVAEIVPSFINKDIETIIIMLEFIANLPPELKAALSERQPSLPELQQYVPALKDSNLSFEEFKKLIRNRQSEAADSNPSELKYLGLDTHSQKKRRPYVALFEKCCLIGCTKRSLAKYC

SEQ ID NO: 5 >gi1119579171Igb1EAW58767.1I relaxin 1, isoform CRA_a [Homo sapiens]MPRLFLFHLLEFCLLLNQFSRAVAAKWKDDVIKLCGRELVRAQIAICGMSTWSKRSLSQEDAPQTPRPVA EIVPSFINKDIETIIIMLEFIANLPPELKAALSERQPSLPELQQYVPALKDSNLSFEEFKKLIRNRQSEAADSNPSELKYLGLDTHSQKKRRPYVALFEKCCLIGCTKRSLAKYC

SEQ ID NO: 6 >gi119579172gbEAW58768.1 relaxin 1, isoform CRA b [Homo sapiens]MPRLFLFHLLEFCLLLNQFSRAVAAKWKDDVIKLCGRELVRAQIAICGMSTWSKRSLSQEDAPQTPRPVAGISSSLLRRRLFEDHDGPSFLV

SEQ ID NO: 7>gi1119579173Hg⁻bEAW58769.1 relaxin 1, isoform CRA c [Homo sapiens]MLEFIANLPPELKAALSERQPSLPELQQYVPALKDSNLSFEEFKKLIRNRQSEAADSNPSELKYLGLDTHSQKKRRPYVALFEKCCLIGCTKRSLAKYC

SEQ ID NO: 8 >gi119604794gbEAW84388.1 relaxin 3 [Homo sapiens]MARYMLLLLLAVWVLTGELWPGAEARAAPYGVRLCGREFIRAVIFTCGGSRWRRSDILAHEAMGDTFPDADADEDSLAGELDEAMGSSEWLALTKSPQAFYRGRPSWQGTPGVLRGSRDVLAGLSSSCCKWGCSKSEISSLC

SEQ ID NO: 9 >gi187954661gbAAI40936.1 Relaxin 3 [Homo sapiens]MARYMLLLLLAVWVLTGELWPGAEARAAPYGVRLCGREFIRAVIFTCGGSRWRRSDILAHEAMGDTFPDADADEDSLAGELDEAMGSSEWLALTKSPQAFYRGRPSWQGTPWLRGSRDVLAGLSSSCCKWGCSKSEISSLC

SEQ ID NO: 10 >gi17484096HgbAAL40345.1AF447451 1 relaxin 3 [Homo sapiens]MARYMLLLLLAVWVLTGELWPGAEARAAPYGVRLCGREFIRAVIFTCGGSRWRRSDILAHEAMGDTFPDADADEDSLAGELDEAMGSSEWLALTKSPQAFYRGRPSWQGTPGVLRGSRDVLAGLSSSCCKWGCSKSEISSLC

SEQ ID NO: 11>gi317373369spJ′51460.2INSL3 HUMAN RecName: Full-Insulin⁻like 3;MDPRLPAWALVLLGPALVFALGPAPTPEMREKLCGHHEVRALVRVCGGPRWSTEARRPATGGDRELLQWLERRHLLHGLVADSNLTLGPGLQPLPQTSHHHRHHRAAATNPARYCCLSGCTQQDLLTLCPY

SEQ ID NO: 12 >gi119579176gbEAW58772.1 insulin-like 4 (placenta) [Homo sapiens]MASLFRSYLPAIWLLLSQLLRESLAAELRGCGPRFGKHLLSYCPMPEKTFTTTPGGWLLESGRPKEMVSTSNNKDGQALGTTSEFIPNLSPELKKPLSEGQPSLKKIILSRKKRSGRHRFDPFCCEVICDDGTSVKLCT

SEQ ID NO: 13 >gi20070773HgbAAH26254.1 Insulin-like 4 (placenta) [Homo sapiens]MASLFRSYLPAIWLLLSQLLRESLAAELRGCGPRFGKHLLSYCPMPEKTFTTTPGGWLLESGRPKEMVSTSNNKDGQALGTTSEFIPNLSPELKKPLSEGQPSLKKIILSRKKRSGRHRFDPFCCEVICDDGTSVKLCT

SEQ ID NO: 14 >gi37183171AQ89389.1 INSL5 [Homo sapiens]MKGSIFTLFLFSVLFAISEVRSKESVRLCGLEYIRTVIYICASSRWRRHLEGIPQAQQAETGNSFQLPHKREFSEENPAQNLPKVDASGEDRLWGGQMPTEELWKSKKHSVMSRQDLQTLCCTDGCSMTDLSALC

SEQ ID NO: 15 >giH4768935gbAAD29686.1AF133816 1 insulin-like peptide INSL5 [Homosapiens]MKGSIFTLFLFSVLFAISEVRSKESVRLCGLEYIRTVIYICASSRWRRHLEGIPQAQQAETGNSFQLPHKREFSEENPAQNLPKVDASGEDRLWGGQMPTEELWKSKKHSVMSRQDLQTLCCTDGCSMTDLSALC

SEQ ID NO: 16 >gik5059419gbAAD39003.1AF156094 1 insulin-like protein 6 [Homo sapiens]      MPRLLRLSLLWLGLLLVRFSRELSDISSARKLCGRYLVKEIEKLCGHANWSQFRFEEETPFSRLIAQASEKVEAYSPYQFESPQTASPARGRGTNPVSTSWEEAVNSWEMQSLPEYKDKKGYSPLGKTREFSSSHNINVYIHENAFFQKKRRNKIKTLSNLFWGHHPQRKRRGYSEKCCLTGCTKEELSIACLPYIDFKRLKEKRSSLVTKIY

1. A formulation comprising microparticles comprising an aliphaticpolyester and an antifibrotic agent, wherein (i) said microparticleshave a diameter of 1-100 µm; (ii) the antifibrotic agent is relaxin andis present in an amount that is 0.01-10% of total mass; (iii) saidaliphatic polyester is of molecular weight 10,000-200,000 Daltons; or(iv) the microparticles further comprise a vinyl polymer.
 2. Theformulation of claim 1, wherein said antifibrotic agent is an agonist ofthe receptor RXFP1.
 3. The formulation of claim 1, wherein saidantifibrotic agent is human relaxin-2 or an analog or variant.
 4. Theformulation of claim 1, wherein the aliphatic polyester ispoly-lactide-co-glycolide or polycaprolactone.
 5. The formulation ofclaim 1, wherein the aliphatic polyester is polycaprolactone.
 6. Theformulation of claim 1, wherein said aliphatic polyester is terminatedby an ester functional group, an alkyl-ester functional group or acarboxylic acid functional group.
 7. The formulation of claim 1, whereinsaid formulation comprises a vinyl polymer that is poly(vinyl alcohol)or poly(pyrrolidone).
 8. The formulation of claim 1, wherein saidformulation comprises a vinyl polymer that is of molecular weight10,000-200,000 Daltons.
 9. The formulation of claim 1, wherein thediameter of said microparticles is 1-75 µm; or 1-50 µm; or 5-50 µm; or25-50 µm; or 30-50 µm; or 40-50 µm.
 10. The formulation of claim 1,wherein said aliphatic polyester is poly-lactide-co-glycolide with amolar ratio of 15:85 - 25:75, lactide:glycolide.
 11. The formulation ofclaim 1, wherein the formulation comprises the vinyl polymer in anamount of 0.01-0.1% of total mass.
 12. The formulation of claim 1,wherein said antifibrotic agent is 0.005-5% of the total formulationmass.
 13. The formulation of claim 1, wherein said antifibrotic agent isa relaxin.
 14. The formulation of claim 1, wherein said formulationcomprises microparticles suspended in a vehicle solution.
 15. Theformulation of claim 1, wherein said formulation comprisesmicroparticles suspended in a sodium chloride liquid solution or asodium carboxymethylcellulose solution.
 16. The formulation of claim 1,wherein said formulation comprises microparticles in lyophilized powderform.
 17. The formulation of claim 1, wherein said formulation is asustained release formulation.
 18. The formulation of claim 1, whereinsaid formulation is a sustained release formulation and wherein theantifibrotic agent is released over an extended period of time.
 19. Theformulation of claim 1, wherein said formulation is a sustained releaseformulation wherein the antifibrotic agent is released over an extendedperiod of least 1 day; or at least 2 days; or at least 3 days; or atleast 4 days; or at least 5 days; or at least 6 days; or at least 1week; or at least 2 weeks; or at least 3 weeks; or at least 4 weeks; orat least 5 weeks, or at least 6 weeks; or at least 8 weeks; or at least9 weeks; at least 10 weeks; or at least 12 weeks; or at least 15 weeks;or between 1-5 days; or between 2-5 days; or between 1-2 days; orbetween 2-3 days; or between 3-4 days; or between 4-5 days; or between3-10 days; or between 1-15 weeks; or between 2-10 weeks; or between 4-8weeks; or between 8-15 weeks; or about 1 day; or about 2 days; or about3 days; or about 4 days; or about 5 days; or about 6 days; or about 1week; or about 2 weeks; or about 3 weeks; or about 4 weeks; or about 5weeks; or about 6 weeks; or about 7 weeks; or about 8 weeks; or about 9weeks; or about 10 weeks, or more.
 20. The formulation of claim 1,wherein the formulation is formulated for administration via inhalationas an aerosol, administration via intra-articular injection oradministration via intramuscular injection.
 21. The formulation of claim1, wherein the formulation is administered to the subject such that theantifibrotic agent is administered to a subject at a dose between 1-2000µg/kg body weight.
 22. A method of treating a fibrotic disease, themethod comprising administering to a subject in need thereof aformulation of claim
 1. 23. A method, said method comprising identifyinga subject diagnosed with one or more diseases selected from the group ofdiseases listed in Table 1 or Table 2 and administering a formulation ofclaim 1 to the subject.
 24. The method of claim 22, wherein the diseaseis selected from the group consisting of Duchenne Muscular Dystrophy ,Becker Muscular Dystrophy , stroke, traumatic brain injury, peripheralnerve injury, Spinal Muscular Atrophy (Type I, II, III, or IV), CerebralPalsy, Arthrogryposis Multiplex Congenita, fibrosis of thehumeroradialjoint, fibrosis of the humeroulnar joint, fibrosis of theglenohumeraljoint, fibrosis of the tibiofemoraljoint, fibrosis of theacetabulofemoral joint, fibrosis of the talocrural joint, fibrosis ofthe temporomandibular joint, fibrosis of the metacarpophalangeal joint,fibrosis of the metatarsophalangeal joint, fibrosis of theperi-articular musculature, cellulite and interstitial lung disease. 25.The method of claim 22, wherein said administering is via inhalation asan aerosol, via intra-articular injection, via intramuscular injection,via intradermal injection., via subcutaneous injection, viaintracapsular injection, via pericapsular injection, viamusculotendinous injection, via intraligamentous injection, viaperiligamentous injection, via intratendinous injection, viaperitendinous injection, via intraosteotendinous injection, viaperiosteotendinous injection.
 26. The method of claim 22, wherein thedisease is Duchene’s muscular dystrophy and said administering is viaintramuscular injection; the disease is Duchene’s muscular dystrophyandsaid administering is via intraarticular injection; the disease isBecker’s muscular dystrophy and said administering is via intramuscularinjection; the disease is Becker’s muscular dystrophy and saidadministering is via intraarticular injection; the disease is SpinalMuscular Dystrophy and said administering is via intramuscularinjection; the disease is Spinal Muscular Dystrophy and saidadministering is via intraarticular injection; the disease isArthrogryposis Multiplex Congenita and said administering is viaintramuscular injection; the disease is Arthrogryposis MultiplexCongenita and said administering is via intraarticular injection; thedisease is Cerebral Palsy and said administering is via intramuscularinjection; the disease is Cerebral Palsy and said administering is viaintraarticular injection; the disease is stroke and said administeringis via intramuscular injection; the disease is stroke and saidadministering is via intraarticular injection; the disease is traumaticbrain injury and said administering is via intramuscular injection; thedisease is traumatic brain injury and said administering is viaintraarticular injection; the disease is peripheral nerve injury andsaid administering is via intramuscular injection; the disease isperipheral nerve injury and said administering is via intraarticularinjection.
 27. The method of claims 22, wherein microparticles are ofsizes between 1um-10um and said administering is via inhalation as anaerosol; the microparticles are of sizes between 20um-100um and saidadministering is via intramuscular injection; the microparticles are ofsizes between 5um-50 um and said administering is via intraarticularinjection.
 28. The method of claim 22, wherein the disease isinterstitial lung disease, the diameter of the microparticle is 1-10 um,and said administering is via inhalation as an aerosol; the disease isDuchene’s Muscular Dystrophy, the diameter of the microparticle is 10-30um, and said administering is via intraarticular injection; the diseaseis Duchene’s Muscular Dystrophy, the diameter of the microparticle is25-50 um, and said administering is via intraarticular injection; thedisease is Duchene’s Muscular Dystrophy, the diameter of themicroparticle is 10-30 um, and said administering is via intramuscularinjection; the disease is Duchene’s Muscular Dystrophy, the diameter ofthe microparticle is 25-50 um, and said administering is viaintramuscular injection; the disease is Spinal Muscular Atrophy, thediameter of the microparticle is 10-30 um, and said administering is viaintraarticular injection; the disease is Spinal Muscular Atrophy, thediameter of the microparticle is 25-50 um, and said administering is viaintraarticular injection; the disease is Spinal Muscular Atrophy, thediameter of the microparticle is 10-30 um, and said administering is viaintramuscular injection; the disease is Spinal Muscular Atrophy, thediameter of the microparticle is 25-50 um, and said administering is viaintramuscular injection; the disease is joint arthrofibrosis, thediameter of the microparticle is 10-30 um, and said administering is viaintraarticular injection; the disease is joint arthrofibrosis, thediameter of the microparticle is 25-50 um, and said administering is viaintraarticular injection.
 29. The method of claim 22, wherein theformulation is administered to the subject such that the antifibroticagent is administered to a subject at a dose between 1-2000 µg/kg bodyweight.